Human AML1/MDS1/EVI1 fusion protein induces an acute myelogenous leukemia (AML) in mice: a model for human AML.
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The human t(3;21)(q26;q22) translocation is found as a secondary mutation in some cases of chronic myelogenous leukemia during the blast phase and in therapy-related myelodysplasia and acute myelogenous leukemia. One result of this translocation is a fusion between the AML1, MDS1, and EVI1 genes, which encodes a transcription factor of approximately 200 kDa. The role of the AML1/MDS1/EVI1 (AME) fusion gene in leukemogenesis is largely unknown. In this study, we analyzed the effect of the AME fusion gene in vivo by expressing it in mouse bone marrow cells via retroviral transduction. We found that mice transplanted with AME-transduced bone marrow cells suffered from an acute myelogenous leukemia (AML) 5-13 mo after transplantation. The disease could be readily transferred into secondary recipients with a much shorter latency. Morphological analysis of peripheral blood and bone marrow smears demonstrated the presence of myeloid blast cells and differentiated but immature cells of both myelocytic and monocytic lineages. Cytochemical and flow cytometric analysis confirmed that these mice had a disease similar to the human acute myelomonocytic leukemia. This murine model for AME-induced AML will help dissect the molecular mechanism of AML and the molecular biology of the AML1, MDS1, and EVI1 genes. (+info)
Transcription-coupled translation control of AML1/RUNX1 is mediated by cap- and internal ribosome entry site-dependent mechanisms.
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AML1/RUNX1 belongs to the runt domain transcription factors that are important regulators of hematopoiesis and osteogenesis. Expression of AML1 is regulated at the level of transcription by two promoters, distal (D) and proximal (P), that give rise to mRNAs bearing two distinct 5' untranslated regions (5'UTRs) (D-UTR and P-UTR). Here we show that these 5'UTRs act as translation regulators in vivo. AML1 mRNAs bearing the uncommonly long (1,631-bp) P-UTR are poorly translated, whereas those with the shorter (452-bp) D-UTR are readily translated. The low translational efficiency of the P-UTR is attributed to its length and the cis-acting elements along it. Transfections and in vitro assays with bicistronic constructs demonstrate that the D-UTR mediates cap-dependent translation whereas the P-UTR mediates cap-independent translation and contains a functional internal ribosome entry site (IRES). The IRES-containing bicistronic constructs are more active in hematopoietic cell lines that normally express the P-UTR-containing mRNAs. Furthermore, we show that the IRES-dependent translation increases during megakaryocytic differentiation but not during erythroid differentiation, of K562 cells. These results strongly suggest that the function of the P-UTR IRES-dependent translation in vivo is to tightly regulate the translation of AML1 mRNAs. The data show that AML1 expression is regulated through usage of alternative promoters coupled with IRES-mediated translation control. This IRES-mediated translation regulation adds an important new dimension to the fine-tuned control of AML1 expression. (+info)
The AML1 transcription factor functions to develop and maintain hematogenic precursor cells in the embryonic aorta-gonad-mesonephros region.
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We examined the role of the AML1 transcription factor in the development of hematopoiesis in the paraaortic splanchnopleural (P-Sp) and the aorta-gonad-mesonephros (AGM) regions of mouse embryos. The activity of colony-forming units of colonies from the P-Sp/AGM region was reduced severalfold by heterozygous disruption of the AML1 gene, indicating that AML1 functioned in a dosage-dependent manner to generate hematopoietic progenitors. In addition, no hematopoietic progenitor activity was detected in the P-Sp/AGM region of embryos with an AML1 null mutation. Similar results were obtained when a dispersed culture was first prepared from the P-Sp/AGM region before assay of the activity of the colony-forming units. In a culture of cells with the AML1(+/+) genotype, both hematopoietic and endothelial-like cell types emerged, but in a culture of cells with the AML1(-/-) genotype, only endothelial-like cells emerged. Interestingly, introduction of AML1 cDNA into the P-Sp/AGM culture with the AML1(-/-) genotype partially restored the production of hematopoietic cells. This restoration was observed for cultures prepared from 9.5-day postcoitum (dpc) embryos but not for cultures prepared from 11.5-dpc embryos. Therefore, the population of endothelial-like cells capable of growing in the AML1(-/-) culture would appear to contain inert but nonetheless competent hematogenic precursor cells up until at least the 9.5-dpc period. All these results support the notion that the AML1 transcription factor functions to develop and maintain hematogenic precursor cells in the embryonic P-Sp/AGM region. (+info)
Biophysical characterization of interactions between the core binding factor alpha and beta subunits and DNA.
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Core binding factors (CBFs) play key roles in several developmental pathways and in human disease. CBFs consist of a DNA binding CBFalpha subunit and a non-DNA binding CBFbeta subunit that increases the affinity of CBFalpha for DNA. We performed sedimentation equilibrium analyses to unequivocally establish the stoichiometry of the CBFalpha:beta:DNA complex. Dissociation constants for all four equilibria involving the CBFalpha Runt domain, CBFbeta, and DNA were defined. Conformational changes associated with interactions between CBFalpha, CBFbeta, and DNA were monitored by nuclear magnetic resonance and circular dichroism spectroscopy. The data suggest that CBFbeta 'locks in' a high affinity DNA binding conformation of the CBFalpha Runt domain. (+info)
Nitric oxide reduces vascular smooth muscle cell elastase activity through cGMP-mediated suppression of ERK phosphorylation and AML1B nuclear partitioning.
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Nitric oxide (NO) reduces the severity of pulmonary vascular disease in rats as do elastase inhibitors. We therefore hypothesized that NO inhibits elastase by suppressing mitogen-activated protein kinases that trans-activate AML1B, a transcription factor for elastase. We used cultured pulmonary artery smooth muscle cells in which serum-treated elastin (STE) induces a > threefold increase in elastase activity as evaluated by solubilization of [(3)H]-elastin. NO donors (SNAP and DETA NONOate) inhibited elastase in a dose-dependent manner as did a cGMP mimetic (8-pCPT-cGMP). SNAP inhibition of elastase was reversed by coadministration of a cGMP-PKG inhibitor (Rp-8-pCPT-cGMP). The STE-induced increase in phospho-ERK was suppressed by NO donors and the cGMP mimetic, and reversed by cGMP-PKG inhibitor, as was expression of AML1B and DNA binding in nuclear extracts. A concomitant increase in p38 phosphorylation was also inhibited by SNAP, but whereas MEK inhibitor (PD98059) suppressed elastase and AML1B-DNA binding, a p38 inhibitor (SB202190) did not. Our study uniquely links NO with inhibition of elastase-dependent matrix remodeling in vascular disease by suggesting a cGMP-PKG-related mechanism suppressing ERK-mediated partitioning of AML1B in nuclear extracts. (+info)
AML1 gene amplification: a novel finding in childhood acute lymphoblastic leukemia.
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BACKGROUND AND OBJECTIVE: We previously found a high-level amplification in chromosomal region 21q22 in two children with acute lymphoblastic leukemia (ALL) using comparative genomic hybridization. The same region harbors the AML1 gene. The aim of the present study was to investigate whether AML1 is a target gene in these amplifications. DESIGN AND METHODS: Bone marrow samples were obtained from 112 childhood ALL patients. The copy number of AML1 was studied using fluorescent in situ hybridization with a dual color DNA probe specific for the AML1 and TEL genes. RESULTS: Three of the patients had 3-to-8 fold amplification of AML1 and showed a high-level amplification of 21q22 by comparative genomic hybridization. In two of them the extra copies were shown to be located tandemly in a derivative of chromosome 21. Thirty-seven of the patients (33%) had 1-to-2 extra copies of AML1, most probably reflecting the incidence of trisomy 21 and tetrasomy 21. The TEL-AML1 fusion was less frequent in the patients with extra copies of AML1 (7/40; 18%) than in the patients with no extra copy (24/72; 33%). None of the three patients with 3-to-8 fold amplification of AML1 showed the fusion or loss of TEL. INTERPRETATION AND CONCLUSIONS: Our findings suggest that the AML1 gene is a target gene in the 21q22 amplicon in childhood ALL. To understand the role, if any, of the AML1 amplification in leukemogenesis, further studies are needed. (+info)
Molecular detection of TEL-AML1 transcripts as a diagnostic tool and for monitoring of minimal residual disease in B-lineage childhood acute lymphoblastic leukemia.
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The chromosomal translocation t(12;21) (p12;q22) which results in the TEL-AML1 fusion gene is the most frequent genetic rearrangement in childhood B-lineage acute lymphoblastic leukemia (ALL). The rearrangement in this locus, however, is only rarely observed by routine karyotypic analysis. We established a nested-reverse transcriptase-polymerase chain reaction (nested-RT-PCR) technique for the detection of the TEL-AML1 transcript, and used this to investigate the incidence of the rearrangement, and to characterize the disease present in TEL-AML1-positive B-lineage ALL patients. The TEL-AML1 fusion transcript was detected in nine of fourteen patients. These patients were relatively homogeneous in that they were young and had low presenting leukocyte counts, both features of which are associated with a favorable prognosis. Furthermore, we could detect the TEL-AML1 transcript in the peripheral blood of t(12;21)-positive patients and we used this to assess minimal residual disease (MRD) in patients during chemotherapy. The data demonstrate that nested-RT-PCR is a suitable tool for diagnosing t(12;21)-positive ALL, that these patients constitute a clinically distinct subgroup of ALL patients, and that the method could also be used to monitor MRD in these patients. (+info)
A novel syndrome of radiation-associated acute myeloid leukemia involving AML1 gene translocations.
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AML1 is a transcriptional activator that is essential for normal hematopoietic development. It is the most frequent target for translocations in acute leukemia. We recently identified 3 patients in whom pancytopenia developed almost 50 years after high-level radiation exposure from nuclear explosions during or after World War II. In all 3 patients, acute myeloid leukemia (AML) eventually developed that had similar characteristics and clinical courses. Cytogenetics from the 3 patients revealed a t(1;21)(p36;q22), a t(18;21)(q21;q22), and a t(19;21)(q13.4;q22). By fluorescent in situ hybridization (FISH), all 3 translocations disrupted the AML1 gene. Two of these AML1 translocations, the t(18;21) and the t(19;21), have not been reported previously. It is possible that the AML1 gene is a target for radiation-induced AML. (Blood. 2000;95:4011-4013) (+info)