Analysis of the role of AML1-ETO in leukemogenesis, using an inducible transgenic mouse model.

As reported previously, AML1-ETO knock-in mice were generated to investigate the role of AML1-ETO in leukemogenesis and to mimic the progression of t(8;21) leukemia. These knock-in mice died in midgestation because of hemorrhaging in the central nervous system and a block of definitive hematopoiesis during embryogenesis. Therefore, they are not a good model system for the development of acute myeloid leukemia. Therefore, mice were generated in which the expression of AML1-ETO is under the control of a tetracycline-inducible system. Multiple lines of transgenic mice have been produced with the AML1-ETO complementary DNA controlled by a tetracycline-responsive element. In the absence of the antibiotic tetracycline, AML1-ETO is strongly expressed in the bone marrow of AML1-ETO and tet-controlled transcriptional activator double-positive transgenic mice. Furthermore, the addition of tetracycline reduces AML1-ETO expression in double-positive mice to nondetectable levels. Throughout the normal murine lifespan of 24 months, mice expressing AML1-ETO have not developed leukemia. In spite of this, abnormal maturation and proliferation of progenitor cells have been observed from these animals. These results demonstrate that AML1-ETO has a very restricted capacity to transform cells. Either the introduction of additional genetic changes or the expression of AML1-ETO at a particular stage of hematopoietic cell differentiation will be necessary to develop a model for studying the pathogenesis of t(8;21).

Synergistic up-regulation of the myeloid-specific promoter for the macrophage colony-stimulating factor receptor by AML1 and the t(8;21) fusion protein may contribute to leukemogenesis.

AML1 is involved in the (8;21) translocation, associated with acute myelogenous leukemia (AML)-type M2, which results in the production of the AML1-ETO fusion protein: the amino-terminal 177 amino acids of AML1 and the carboxyl-terminal 575 amino acids of ETO. The mechanism by which AML1-ETO accomplishes leukemic transformation is unknown; however, AML1-ETO interferes with AML1 transactivation of such AML1 targets as the T-cell receptor beta enhancer and the granulocyte-macrophage colony-stimulating factor promoter. Herein, we explored the effect of AML1-ETO on regulation of a myeloid-specific AML1 target, the macrophage colony-stimulating factor (M-CSF) receptor promoter. We found that AML1-ETO and AML1 work synergistically to transactivate the M-CSF receptor promoter, thus exhibiting a different activity than previously described. Truncation mutants within the ETO portion of AML1-ETO revealed the region of ETO necessary for the cooperativity between AML1 and AML1-ETO lies between amino acids 347 and 540. Endogenous M-CSF receptor expression was examined in Kasumi-1 cells, derived from a patient with AML-M2 t(8;21) and the promonocytic cell line U937. Kasumi-1 cells exhibited a significantly higher level of M-CSF receptor expression than U937 cells. Bone marrow from patients with AML-M2 t(8;21) also exhibited a higher level of expression of M-CSF receptor compared with normal controls. The upregulation of M-CSF receptor expression by AML1-ETO may contribute to the development of a leukemic state in these patients.

CCAAT enhancer-binding protein (C/EBP) and AML1 (CBF alpha2) synergistically activate the macrophage colony-stimulating factor receptor promoter.

Transcription factors play a key role in the development and differentiation of specific lineages from multipotential progenitors. Identification of these regulators and determining the mechanism of how they activate their target genes are important for understanding normal development of monocytes and macrophages and the pathogenesis of a common form of adult acute leukemia, in which the differentiation of monocytic cells is blocked. Our previous work has shown that the monocyte-specific expression of the macrophage colony-stimulating factor (M-CSF) receptor is regulated by three transcription factors interacting with critical regions of the M-CSF receptor promoter, including PU.1 and AML1.PU.1 is essential for myeloid cell development, while the AML1 gene is involved in several common leukemia-related chromosome translocations, although its role in hematopoiesis has not been fully identified. Along with AML1, a third factor, Mono A, interacts with a small region of the promoter which can function as a monocyte-specific enhancer when multimerized and linked to a heterologous basal promoter. Here, we demonstrate by electrophoretic mobility shift assays with monocytic nuclear extracts, COS-7 cell-transfected factors, and specific antibodies that the monocyte-enriched factor Mono A is CCAAT enhancer-binding protein (C/EBP). C/EBP has been shown previously to be an important transcription factor involved in hepatocyte and adipocyte differentiation; in hematopoietic cells, C/EBP is specifically expressed in myeloid cells. In vitro binding analysis reveals a physical interaction between C/EBP and AML1. Further transfection studies show that C/EBP and AML1 in concert with the AML1 heterodimer partner CBF beta synergistically activate M-CSF receptor by more then 60 fold. These results demonstrate that C/EBP and AML1 are important factors for regulating a critical hematopoietic growth factor receptor, the M-CSF receptor, suggesting a mechanism of how the AML1 fusion protein could contribute to acute myeloid leukemia. Furthermore, they demonstrate physical and functional interactions between AML1 and C/EBP transcription factor family members.

The adenoviral transcription factor, E1A 13S, trans-activates the human tumor necrosis factor-alpha promoter.

The 1311 bp TNF-alpha promoter region fused to a luciferase reporter vector was used in a transient transfection system to study the regulation of TNF-alpha promoter activity by E1A 13S in the U937 macrophage cell line and the MLA 144 T cell line. Co-transfections of the TNF-alpha promoter with an E1A expression vector resulted in a strong trans-activation of the promoter in both cell lines. Sequential truncation of the promoter mapped the E1A responsive region to sequences contained between -120 bp and the transcription start site. Truncation to -95 bp caused a dramatic 87% reduction of E1A activation in MLA 144 cells and further truncation to -36 bp caused a complete loss of E1A activation. In U937 cells, each truncation lowered E1A responsiveness but activity was never completely abolished. Site-directed mutagenesis of putative cis-acting sequences in the TNF-alpha promoter identified the AP-1 site as important for E1A trans-activation in the U937 cell line; the AP-2 and CRE sites also appeared to contribute to a lesser degree. In contrast, only the CRE mutation caused a reduction in E1A induced activity in the MLA 144 cell line. Co-transfection of the E1A expression vector with expression vectors for the cellular transcription factors AP-1, AP-2 and CREB indicated that none of these transcription factors showed any co-operativity with E1A. Thus, cis-acting sequences which contribute to E1A trans-activation of the TNF-alpha promoter have been delineated.

Granulocyte-macrophage colony-stimulating factor and interleukin-4 differentially regulate the human tumor necrosis factor-alpha promoter region.

Regulation of TNF-alpha promoter activity by IL-2, IFN-gamma, GM-CSF, and IL-4 was examined in the U937 macrophage cell line and the MLA 144 T cell line. Using a transient transfection system, the full-length TNF-alpha promoter was examined for response to these cytokine signals. Only GM-CSF was able to consistently induce a twofold activation of the TNF-alpha promoter in the U937 cell line. GM-CSF activation of the promoter region was further analyzed using a series of 5' truncations and site mutations of the AP-1, AP-2, and CRE sites of the promoter. The GM-CSF activation mapped to the region contained within the 95 base pairs upstream from the transcription start site (TSS) with the AP-2 site as a putative cis-acting sequence. IL-4 profoundly inhibited both basal and phorbol ester-induced TNF-alpha promoter activity as well as protein production. Promoter inhibition by IL-4 required the 95-bp basal promoter sequence.

The regulation of the human tumor necrosis factor alpha promoter region in macrophage, T cell, and B cell lines.

The 1311-base pair human tumor necrosis factor (TNF) alpha promoter region was fused to the luciferase (Luc) reporter gene and studied in a transient transfection system in three TNF producing cell lines, the U937 macrophage cell line, the MLA 144 T cell line, and the 729-6 B cell line. This full length promoter construct can be induced by phorbol 13-myristate acetate (PMA) in each of these cell types. Analysis of a series of 5'-truncations showed several peaks of basal and PMA induced activity suggesting the presence of several positive and negative regulatory elements. A PMA responsive element was localized to a region between -95 and -36 bp relative to the transcription start site. Within this region, single AP-2- and AP-1-like consensus sequences were noted. These AP-2 and AP-1 sites were each modified with a double point mutation. A modest (20-50%) reduction in TNF promoter activity was observed with the AP-2 site mutation. However, mutation of the AP-1 site markedly diminished both the basal and PMA-activated promoter activity. Also co-transfections of the wild-type promoter construct with an AP-1/c-jun expression vector resulted in augmented basal and PMA-induced promoter activity.