Transcriptional activation of erythropoietin, glycolytic enzymes, and vascular endothelial growth factor occurs during hypoxia or in response to cobalt chloride (CoCl 2 ) in Hep3B cells. However, neither the mechanism of cellular O 2 sensing nor that of cobalt is fully understood. We tested whether mitochondria act as O 2 sensors during hypoxia and whether hypoxia and cobalt activate transcription by increasing generation of reactive oxygen species (ROS). Results show (i) wild-type Hep3B cells increase ROS generation during hypoxia (1.5% O 2 ) or CoCl 2 incubation, (ii) Hep3B cells depleted of mitochondrial DNA ( 0 cells) fail to respire, fail to activate mRNA for erythropoietin, glycolytic enzymes, or vascular endothelial growth factor during hypoxia, and fail to increase ROS generation during hypoxia; (iii) 0 cells increase ROS generation in response to CoCl 2 and retain the ability to induce expression of these genes; and (iv) the antioxidants pyrrolidine dithiocarbamate and ebselen abolish transcriptional activation of these genes during hypoxia or CoCl 2 in wild-type cells, and abolish the response to CoCl 2 in °cells. Thus, hypoxia activates transcription via a mitochondria-dependent signaling process involving increased ROS, whereas CoCl 2 activates transcription by stimulating ROS generation via a mitochondria-independent mechanism.

The EVII gene, located at chromosome band 3q26, is overexpressed in some myeloid leukemia patients with breakpoints either 5' of the gene in the t(3;3)(q21;q26) or 3' of the gene in the inv(3)(q21q26). EVIl is also expressed as part of a fusion transcript with the transcription factorAMLI in the t(3;21)(q26;q22), associated with myeloid leukemia. In cells with t(3;21), additional fusion transcripts are AMLIMDSl andAMLI-MDSI-EV71.MDSJ is located at 3q26 170-400 kb upstream (telomeric) The protooncogene EVI1 was initially identified and described in the mouse. It is activated in murine myeloid leukemia by proviral insertion in the evil common integration site (1, 2). EVIl is not normally expressed in hematopoietic cells. In humans, the gene can be activated in myeloid leukemias and myelodysplastic diseases by chromosomal rearrangements either 5' of the gene in the t(3;3)(q21;q26) or 3' of the gene in the inv(3)(q21q26) by juxtaposition of the gene to enhancer elements of the ribophorin gene located at 3q21 (3-5). Activation of EVIl can also occur in the t(3;21)(q26;q22) as part of the fusion mRNA,AMLl-EV71, that is transcribed from the der(3) chromosome (6, 7). Abnormal expression of EVI1 has also been detected in patients with myeloid leukemia and a cytogenetically normal karyotype (8), suggesting that inappropriate activation of this gene occurs through various mechanisms.EV71 is a nuclear protein containing a seven-zinc-finger domain at the N-terminal end, a three-finger domain in the central part of the molecule, and an acidic domain distal to the second group of zinc fingers (9). The human and mouse open reading frames are 91% homologous at the DNA level and 94% homologous at the amino acid level. The second exon of the gene, in frame although not translated, is highly conserved between the two species. The open reading frame starts in the third exon of the gene, where the first ATG is located (9). A The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact. putative promoter has been identified, by genomic sequencing and Si protection analysis, immediately upstream of the first exon of the murine cDNA (10).MDS1 was cloned as one of the partner genes of AML1 in the t(3;21)(q26;q22), associated with therapy-related acute myeloid leukemia and myelodysplastic syndrome as well as with chronic myeloid leukemia in blast crisis (11,12). In this translocation, AML1, located at 21q22, is fused to several genes, EAP, MDS1, and EVIl, all of which are located at 3q26200-400 kb apart (7), and chimeric cDNAs have been isolated from cells with t(3;21) in which AML1 is fused to EAP, to MDS1, to EVI1, or to MDS1 and EVIl in the same transcript, producing, in the latter case, a very complex chimeric gene. Only the 3' region of MDS1 that is fused to AML1 has been isolated and sequenced. The nature of MDS1 is somewhat controversial; MDS1 has been described as a unique ge...

Mechanical strain triggers a variety of cellular responses, but the underlying mechanotransduction process has not been established. Endothelial cells (EC) respond to mechanical strain by upregulating adhesion molecule expression through a signaling process involving reactive oxygen species (ROS), but the site of their generation is unknown. Mitochondria anchor to the cytoskeleton and could function as mechanotransducers by releasing ROS during cytoskeletal strain. In human umbilical vein EC (HUVEC), ROS production increased 221 +/- 17% during 6 h of cyclic strain vs. unstrained controls. Mitochondrial inhibitors diphenylene iodonium or rotenone abrogated this response, whereas inhibitors of nitric oxide (NO) synthase (L-nitroarginine), xanthine oxidase (allopurinol), or NAD(P)H oxidase (apocynin) had no effect. The antioxidants ebselen and diethyldithiocarbamate inhibited the increase in ROS, but the NO scavenger Hb had no effect. Thus strain induces ROS release from mitochondria. In other studies, HUVEC were rendered mitochondria deficient (rho0 EC) to determine the requirement for electron transport in the response to strain. Strain-induced 2'7'-dichlorofluorescein fluorescence was attenuated by >80% in rho0 EC compared with HUVEC (43 +/- 7 vs. 221 +/- 17%). Treatment with cytochalasin D abrogated strain-induced ROS production, indicating a requirement for the actin cytoskeleton. Cyclic strain (6 h) increased VCAM-1 expression in wild-type but not rho0 EC. Increases in NF-kappaB activation and VCAM-1 mRNA expression during strain were prevented by antioxidants. These findings demonstrate that mitochondria function as mechanotransducers in endothelium by increasing ROS signaling, which is required for strain-induced increase in VCAM-1 expression via NF-kappaB.

The (3;21) (q26;q22) translocation associated with treatment-related myelodysplastic syndrome, treatmentrelated acute myeloid leukemia, and blast crisis of chronic myeloid leukemia results in the expression of the chimeric genes AMLI/EAP, AMLl/MDSI, and AMLIE EVIi. AMLI (CBFA2), which codes for the a subunit of the heterodimeric transcription factor CBF, is also involved in the t(8;21), and the gene coding for the 13 subunit (CBFB) is involved in the inv (16 The (3;21)(q26;q22) translocation is a recurring chromosomal abnormality associated with treatment-related acute myeloid leukemia (t-AML), treatment-related myelodysplastic syndrome (t-MDS), and blast crisis of chronic myeloid leukemia (1). The gene on chromosome 21 located at the translocation breakpoint is AML1 (2), which codes for the CBFA2 subunit of the transcription factor CBF. AML1 belongs to a family of CBFA subunits characterized by a 5' region that encodes a domain with both DNA-binding and protein dimerization properties which is homologous to the DNA-binding domain of the Drosophila melanogaster protein encoded by the segmentation gene runt (3, 4). The DNA-binding site of AML1 targets the sequence TGYGGTY (5), which has been identified in regulatory regions of several essential hematopoietic genes (6-8). The runt homology region of AML1 allows binding to CBFB to form a heterodimer that has increasedThe publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact.DNA binding affinity (4). The 3' sequence ofAML1 codes for a transactivation domain (9). AML1 is the human homolog [99% identity of the deduced amino acid (aa) sequence for the first 241 aa residues] of the a subunit of murine Cbf (10). Murine Cbf2 (Pebp2) has been shown to bind and to transactivate the murine myeloperoxidase gene enhancer (6); it is expressed in some T-cell subpopulations in which it may have a role in the regulation of T-cell-specific gene expression (11). AML1 has several isoforms, four of which have been cloned. Two of the isoforms are smaller proteins of 250 aa (2) and 258 aa (1) that consist mainly of the runt homology domain. One of these smaller proteins, AML1A, has been shown to bind the TCRB enhancer without causing transactivation (9). The other two cDNAs encode larger proteins of 472 aa (12) and 479 aa that include a transactivation domain (9). One of them, AML1B, coding for the 479-aa protein induces modest transactivation of the human TCRB (9) and CSF2 (granulocyte/ macrophage-colony-stimulating factor) (7) regulatory regions. Higher levels of transactivation by AMLlB may require the synergistic action of other proteins at the enhancer/promoter site, as demonstrated by the increase in Cbfa2 transactivation of the TCRA enhancer by Ets-1 (13).The CSFIR promoter has binding sites for CBF and PU.1 (a member of the Ets family) (8,14), and CSFIR mutations resulting in ligand-independent upregulated kinase activity ...