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BRIEF REPORT
From the Indiana University Cancer Center,
Indianapolis, Indiana; Cancer Care Consultants, Las Vegas, Nevada;
Molecular and Experimental Medicine, Scripps Research Institute, La
Jolla, California; and Genzyme Genetics, Santa Fe, New Mexico.
AML1 is a transcription factor that is essential for normal
hematopoietic development. It is the most frequent target for translocations in acute leukemia. Recently, fluorescence in situ hybridization was used to identify a novel syndrome of
radiation-associated secondary acute myelogenous leukemia that
had AML1 translocations. Using polymerase chain reaction, the AML1
fusion transcript was isolated from the patient who had a
t(19;21) radiation-associated leukemia. The AML1
gene is fused out of frame to chromosome 19 sequences, resulting in a
truncated AML protein bearing the DNA binding domain but not the
transcriptional activation domain. This fusion AML1 protein
functions as an inhibitor of the normal AML1 protein.
(Blood. 2001;97:2168-2170) The AML1 gene (also known as
RUNX1 and CBFA2) codes for a protein (CBF We recently described data on 3 patients with secondary acute
myelogenous leukemia (AML) after close exposure to nuclear explosions that had translocations in the AML1 gene by fluorescence in
situ hybridization (FISH).8 One of the patients had a
t(19;21)(q13;q22) clonal cytogenetic abnormality involving
AML1. Using rapid amplification of complementary DNA (cDNA)
ends (RACE) polymerase chain reaction (PCR) the AML1 fusion
transcript from this translocation was isolated.
RACE PCR
The AML1 specific primer no. 1 was
5'-ctggccgaccacccgggcgagctggtgcgc, and AML1 specific primer
no. 2 was 5'-caacaagaccctgcccatcgctttcaaggt. Both nested amplifications
took place at 95°C for 2 minutes for 1 cycle, followed by 95°C for
30 seconds and 68°C for 3 minutes for 35 cycles using Advantage 2 Taq
polymerase (Clontech, Palo Alto, CA). The AUAP 3' tail primer was added
after the 10th cycle at 95°C. PCR product subclones were screened by
Southern analysis using radiolabeled AML1 specific primer no. 3:
5'-aggtttgtcggtcgaagtggaagagggaaa. Once the fusion transcript was
identified, the entire coding sequence was amplified using the
following primers, 5'-ttgttgtgatgcgtatccccgtagatgcca and
5'-gtataaattgggactttatttattttc.
Cotransfection assays
Immunohistology Cellular localization of the fusion AML1 protein was performed using AML1 polyclonal antisera (Calbiochem) and immunohistology as we described11 on transiently transfected 293 cells.
Using RACE PCR fusion transcripts from a t(19;21) leukemia that
had a translocated AML1 were isolated and characterized
(Figure 1A). In the most common
transcript, the 5' end of the AML1 gene, after exon 5, containing the DNA binding domain, was fused to a 236-nt sequence
on chromosome 19 DNA termed AMP-19 (Figure 1B). However, rare splice variants had breakpoint junctions after exon 6 of
AML1, indicating that the translocation occurred in intron 6.
Although the two chromosome 19 nucleotide segments (4423 nt apart) that make up AMP19 have classic exon-intron sequences at their boundaries, a BLAST search of the EST database, Northern analysis, hybridization screening of 4 cDNA libraries, and PCR screening of 18 cDNA libraries did not reveal an expressed gene. There is an EST (AI204456) on chromosome 19 within 51 nt of the AMP19 sequence. However, isolating and sequencing the 3.2-kB gene this EST represents revealed that AMP19 is located in opposite orientation within an intron of this EST, and the EST exons around AMP19 are 3' untranslated sequences, making it unlikely that this EST is part of AMP19. Multiple 5' RACE analyses and screening a PCR constructed marrow cDNA library did not reveal a reciprocal der21 messenger RNA (mRNA) product. The untranslocated AML1 transcript was amplified using reverse transcription-PCR,12 subcloned, and sequenced. There were no changes in the amino acid sequence. AML1 is fused out of frame with the AMP19
sequence, which would produce a truncated CBF
We analyzed the cellular location of AML1-AMP19 using immunohistology.11,12 We found that AML1-AMP19 was located both in the cytoplasm and in the nucleus in discrete bundles (Figure 2B,C). The AML1-AMP19 fusion transcript is reminiscent of the AML1-EAP fusion.4 Both are fused out of frame, resulting in truncated AML1 species that function as inhibitory transcription factors.
Submitted July 21, 2000; accepted November 21, 2000.
The publication costs of this article were defrayed in part by page charge payment. Therefore, and solely to indicate this fact, this article is hereby marked "advertisement" in accordance with 18 U.S.C. section 1734.
Reprints: Robert Hromas, Indiana University Cancer Center, R4-202, 1044 W Walnut St, Indianapolis, IN 46202.
1. Van der Reiden BA, Bloomfield CD, Touw IP, Jansen JH. Acute leukemias with structurally altered core binding factor subunits: (t(8;21), inv(16), and t(12;21). Meeting report. Leukemia. 1997;11:2217[Medline] [Order article via Infotrieve].
2.
Roulston D, Espinosa R, Nucifora G, Larson RA, Le Beau MM, Rowley JD.
CBF
3.
Tenen DG, Hromas R, Licht JD, Zhang DE.
Transcription factors, normal myeloid development, and leukemia.
Blood.
1997;90:489
4.
Zent CS, Mathieu C, Claxton DF, et al.
The chimeric genes AML1/MDS1 and AML1/EAP inhibit AML1B activation at the CSF1R promoter, but only AML1/MDS1 has tumor-promoter properties.
Proc Natl Acad Sci U S A.
1996;93:1044 5. Miyoshi H, Kozu T, Shimizu K, et al. The t(8;21) translocation in acute myeloid leukemia results in the production of an AML1-MTG8 transcript. EMBO J. 1993;12:2715[Medline] [Order article via Infotrieve].
6.
Romana SP, Mauchaffe M, Le Coniat M, et al.
The t(12;21) of acute lymphoblastic leukemia results in a TEL-AML1 fusion gene.
Blood.
1995;85:3662
7.
Gamou T, Kitamura E, Hosoda F, et al.
The partner gene of AML1 in t(16;21) myeloid malignancies is a novel member of the MTG8(ETO) family.
Blood.
1998;91:4028
8.
Hromas R, Shopnick R, Jumean HG, Bowers C, Varella-Garcia M, Richkind K.
A novel syndrome of radiation-associated acute myeloid leukemia involving AML1 gene translocations.
Blood.
2000;95:4011 9. Morris J, Rauscher F III, Davis B, et al. The myeloid zinc finger gene MZF-1 regulates the CD34 promoter in vitro. Blood. 1995;86:3940.
10.
Petrovick MS, Hiebert SW, Friedman AD, Hetherington CJ, Tenen DG, Zhang DE.
Multiple functional domains of AML1: PU.1 and C/EBPalpha synergize with different regions of AML1.
Mol Cell Biol.
1998;18:3915
11.
Hromas R, Orazi A, Neiman RS, et al.
Hematopoietic lineage- and stage-restricted expression of the ETS oncogene family member PU.1.
Blood.
1993;82:2998
12.
Osato M, Asou N, Abdalla E, et al.
Biallelic and heterozygous point mutations in the runt domain of the AML1/PEBP2alphaB gene associated with myeloblastic leukemias.
Blood.
1999;93:1817
© 2001 by The American Society of Hematology.
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  E. M. Chan, E. M. Comer, F. C. Brown, K. E. Richkind, M. L. Holmes, B. H. Chong, R. Shiffman, D.-E. Zhang, M. L. Slovak, C. L. Willman, et al. AML1-FOG2 fusion protein in myelodysplasia Blood, June 1, 2005; 105(11): 4523 - 4526. [Abstract] [Full Text] [PDF]
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 J.-Y. Cho, Y. Akbarali, L. F. Zerbini, X. Gu, J. Boltax, Y. Wang, P. Oettgen, D.-E. Zhang, and T. A. Libermann Isoforms of the Ets Transcription Factor NERF/ELF-2 Physically Interact with AML1 and Mediate Opposing Effects on AML1-mediated Transcription of the B Cell-specific blk Gene J. Biol. Chem., May 7, 2004; 279(19): 19512 - 19522. [Abstract] [Full Text] [PDF]
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| Copyright © 2001 by American Society of Hematology Online ISSN: 1528-0020 | |||||||||
Top
Abstract
Introduction
2)
that complexes with CBF
to form the core binding factor (CBF)
complex. Disruption of CBF is the most common alteration in acute
leukemia.
