Epigenetic silencing of Bcl-2, CEBPA and p14ARF by the AML1-ETO oncoprotein contributing to growth arrest and differentiation block in the U937 cell line

Zhuang,Wen-Yue; Cen,Jian-Nong; Zhao,Yun; Chen,Zi-Xing

doi:10.3892/or.2013.2459

Epigenetic silencing of Bcl-2, CEBPA and p14ARF by the AML1-ETO oncoprotein contributing to growth arrest and differentiation block in the U937 cell line

Authors:
- Wen-Yue Zhuang
- Jian-Nong Cen
- Yun Zhao
- Zi-Xing Chen
View Affiliations

Affiliations: The First Affiliated Hospital, Soochow University, Jiangsu Institute of Hematology, Key Laboratory of Thrombosis and Hemostasis, Ministry of Health, Suzhou, Jiangsu, P.R. China, Cyrus Tang Hematology Center, Medical College, Soochow University, Suzhou, Jiangsu, P.R. China
Published online on: May 14, 2013 https://doi.org/10.3892/or.2013.2459
Pages: 185-192

Metrics: Total Views: 0 (Spandidos Publications: | PMC Statistics: )

Metrics: Total PDF Downloads: 0 (Spandidos Publications: | PMC Statistics: )

Cited By (CrossRef): 0 citations Loading Articles...

Abstract

The AML1‑ETO fusion transcription factor generated by the t(8;21) translocation is considered to deregulate the expression of genes that are crucial for normal differentiation and proliferation of hematopoietic progenitors, resulting in acute myelogenous leukemia by recruiting co-repressor complexes to DNA. To investigate the role of AML1‑ETO in leukemogenesis, we transfected the cloned AML1‑ETO cDNA and expressed the AML1‑ETO protein in U937 myelomonocytic leukemia cells. By focusing on the anti-apoptotic gene Bcl‑2, the key regulator gene of granulocytic differentiation CCAAT/enhancer-binding protein α (CEBPA) and the tumor suppressor gene p14ARF, we found that both AML1‑ETO-expressing cell lines and t(8;21) leukemia samples displayed low levels of these three genes. Chromatin immunoprecipitation assays demonstrated that Bcl-2, CEBPA and p14ARF were direct transcriptional targets of AML1‑ETO. The universal binding of AML1‑ETO to genomic DNA resulted in recruitment of methyl-CpG binding protein 2 (MeCP2), reduction of histone H3 or H4 acetylation and increased trimethylation of histone H3 lysine 9 as well as lysine 27 indicating that AML1‑ETO induced heterochromatic silencing of Bcl-2, CEBPA and p14ARF. These results suggested that the aberrant transcription factor AML1‑ETO epigenetically silenced the function of the Bcl-2, CEBPA and p14ARF genes by inducing repressed chromatin configurations at their promoters through histone modifications.

View Figures

View References

1	Chevallier N, Corcoran CM, Lennon C, et al: ETO protein of t(8;21) AML is a corepressor for Bcl-6 B-cell lymphoma oncoprotein. Blood. 103:1454–1463. 2004. View Article : Google Scholar : PubMed/NCBI
2	Rowley JD: Molecular genetics in acute leukemia. Leukemia. 14:513–517. 2000. View Article : Google Scholar : PubMed/NCBI
3	Maiques-Diaz A, Chou FS, Wunderlich M, et al: Chromatin modifications induced by the AML1-ETO fusion protein reversibly silence its genomic targets through AML1 and Sp1 binding motifs. Leukemia. 26:1329–1337. 2012. View Article : Google Scholar : PubMed/NCBI
4	Klampfer L, Zhang J, Zelenetz AO, Uchida H and Nimer SD: The AML1/ETO fusion protein activates transcription of BCL-2. Proc Natl Acad Sci USA. 93:14059–14064. 1996. View Article : Google Scholar : PubMed/NCBI
5	Pabst T, Mueller BU, Harakawa N, et al: AML1-ETO downregulates the granulocytic differentiation factor C/EBPalpha in t(8;21) myeloid leukemia. Nat Med. 7:444–451. 2001. View Article : Google Scholar : PubMed/NCBI
6	Linggi B, Muller-Tidow C, van de Locht L, et al: The t(8;21) fusion protein, AML1 ETO, specifically represses the transcription of the p14(ARF) tumor suppressor in acute myeloid leukemia. Nat Med. 8:743–750. 2002. View Article : Google Scholar : PubMed/NCBI
7	Kelly PN and Strasser A: The role of Bcl-2 and its pro-survival relatives in tumourigenesis and cancer therapy. Cell Death Differ. 18:1414–1424. 2011. View Article : Google Scholar : PubMed/NCBI
8	Krug U, Ganser A and Koeffler HP: Tumor suppressor genes in normal and malignant hematopoiesis. Oncogene. 21:3475–3495. 2002. View Article : Google Scholar : PubMed/NCBI
9	Ho PA, Alonzo TA, Gerbing RB, et al: Prevalence and prognostic implications of CEBPA mutations in pediatric acute myeloid leukemia (AML): a report from the Children’s Oncology Group. Blood. 113:6558–6566. 2009.PubMed/NCBI
10	Zhang DE, Hetherington CJ, Meyers S, et al: CCAAT enhancer-binding protein (C/EBP) and AML1 (CBF alpha2) synergistically activate the macrophage colony-stimulating factor receptor promoter. Mol Cell Biol. 16:1231–1240. 1996.
11	Sherr CJ and Weber JD: The ARF/p53 pathway. Curr Opin Genet Dev. 10:94–99. 2000. View Article : Google Scholar : PubMed/NCBI
12	Kamijo T, Zindy F, Roussel MF, et al: Tumor suppression at the mouse INK4a locus mediated by the alternative reading frame product p19ARF. Cell. 91:649–659. 1997. View Article : Google Scholar : PubMed/NCBI
13	Kohzaki H, Ito K, Huang G, Wee HJ, Murakami Y and Ito Y: Block of granulocytic differentiation of 32Dcl3 cells by AML1/ETO(MTG8) but not by highly expressed Bcl-2. Oncogene. 18:4055–4062. 1999. View Article : Google Scholar : PubMed/NCBI
14	Shikami M, Miwa H, Nishii K, et al: Low BCL-2 expression in acute leukemia with t(8;21) chromosomal abnormality. Leukemia. 13:358–368. 1999. View Article : Google Scholar : PubMed/NCBI
15	Banker DE, Radich J, Becker A, et al: The t(8;21) translocation is not consistently associated with high Bcl-2 expression in de novo acute myeloid leukemias of adults. Clin Cancer Res. 4:3051–3062. 1998.PubMed/NCBI
16	Burel SA, Harakawa N, Zhou L, Pabst T, Tenen DG and Zhang DE: Dichotomy of AML1-ETO functions: growth arrest versus block of differentiation. Mol Cell Biol. 21:5577–5590. 2001. View Article : Google Scholar : PubMed/NCBI
17	Ruas M and Peters G: The p16INK4a/CDKN2A tumor suppressor and its relatives. Biochim Biophys Acta. 1378:F115–F177. 1998.PubMed/NCBI
18	Taniguchi T, Chikatsu N, Takahashi S, et al: Expression of p16INK4A and p14^ARF in hematological malignancies. Leukemia. 13:1760–1769. 1999. View Article : Google Scholar : PubMed/NCBI
19	Hiebert SW, Reed-Inderbitzin EF, Amann J, Irvin B, Durst K and Linggi B: The t(8;21) fusion protein contacts co-repressors and histone deacetylases to repress the transcription of the p14^ARF tumor suppressor. Blood Cells Mol Dis. 30:177–183. 2003. View Article : Google Scholar : PubMed/NCBI
20	Lu Y, Xu YB, Yuan TT, et al: Inducible expression of AML1-ETO fusion protein endows leukemic cells with susceptibility to extrinsic and intrinsic apoptosis. Leukemia. 20:987–993. 2006. View Article : Google Scholar : PubMed/NCBI
21	Fazi F, Zardo G, Gelmetti V, et al: Heterochromatic gene repression of the retinoic acid pathway in acute myeloid leukemia. Blood. 109:4432–4440. 2007. View Article : Google Scholar : PubMed/NCBI
22	Eberharter A and Becker PB: Histone acetylation: a switch between repressive and permissive chromatin. Second in review series on chromatin dynamics. EMBO Rep. 3:224–229. 2002. View Article : Google Scholar : PubMed/NCBI
23	Kouzarides T: Histone methylation in transcriptional control. Curr Opin Genet Dev. 12:198–209. 2002. View Article : Google Scholar : PubMed/NCBI
24	Jones PA and Baylin SB: The epigenomics of cancer. Cell. 128:683–692. 2007. View Article : Google Scholar : PubMed/NCBI
25	Sharma S, Kelly TK and Jones PA: Epigenetics in cancer. Carcinogenesis. 31:27–36. 2010. View Article : Google Scholar
26	Tada Y, Brena RM, Hackanson B, Morrison C, Otterson GA and Plass C: Epigenetic modulation of tumor suppressor CCAAT/enhancer binding protein alpha activity in lung cancer. J Natl Cancer Inst. 98:396–406. 2006. View Article : Google Scholar : PubMed/NCBI
27	Bennett KL, Hackanson B, Smith LT, et al: Tumor suppressor activity of CCAAT/enhancer binding protein alpha is epigenetically down-regulated in head and neck squamous cell carcinoma. Cancer Res. 67:4657–4664. 2007. View Article : Google Scholar : PubMed/NCBI
28	Hackanson B, Bennett KL, Brena RM, et al: Epigenetic modification of CCAAT/enhancer binding protein alpha expression in acute myeloid leukemia. Cancer Res. 68:3142–3151. 2008. View Article : Google Scholar : PubMed/NCBI
29	Wouters BJ, Jordà MA, Keeshan K, et al: Distinct gene expression profiles of acute myeloid/T-lymphoid leukemia with silenced CEBPA and mutations in NOTCH1. Blood. 110:3706–3714. 2007. View Article : Google Scholar : PubMed/NCBI
30	Kiefer MC, Brauer MJ, Powers VC, et al: Modulation of apoptosis by the widely distributed Bcl-2 homologue Bak. Nature. 374:736–739. 1995. View Article : Google Scholar : PubMed/NCBI
31	Hanada M, Delia D, Aiello A, Stadtmauer E and Reed JC: bcl-2 gene hypomethylation and high-level expression in B-cell chronic lymphocytic leukemia. Blood. 82:1820–1828. 1993.PubMed/NCBI
32	Friedrich MG, Weisenberger DJ, Cheng JC, et al: Detection of methylated apoptosis-associated genes in urine sediments of bladder cancer patients. Clin Cancer Res. 10:7457–7465. 2004. View Article : Google Scholar : PubMed/NCBI
33	Esteller M, Tortola S, Toyota M, et al: Hypermethylation-associated inactivation of p14(ARF) is independent of p16(INK4a) methylation and p53 mutational status. Cancer Res. 60:129–133. 2000.PubMed/NCBI
34	Benanti JA, Wang ML, Myers HE, Robinson KL, Grandori C and Galloway DA: Epigenetic down-regulation of ARF expression is a selection step in immortalization of human fibroblasts by c-Myc. Mol Cancer Res. 5:1181–1189. 2007. View Article : Google Scholar : PubMed/NCBI
35	Hollenbach PW, Nguyen AN, Brady H, et al: A comparison of azacitidine and decitabine activities in acute myeloid leukemia cell lines. PLoS One. 5:e90012010. View Article : Google Scholar : PubMed/NCBI
36	Buchi F, Spinelli E, Masala E, et al: Proteomic analysis identifies differentially expressed proteins in AML1/ETO acute myeloid leukemia cells treated with DNMT inhibitors azacitidine and decitabine. Leuk Res. 36:607–618. 2012. View Article : Google Scholar
37	Zapotocky M, Mejstrikova E, Smetana K, Stary J, Trka J and Starkova J: Valproic acid triggers differentiation and apoptosis in AML1/ETO-positive leukemic cells specifically. Cancer Lett. 319:144–153. 2012. View Article : Google Scholar : PubMed/NCBI