No abstract

t(8;21) and t(16;21) create two fusion proteins, AML-1-ETO and AML-1-MTG16, respectively, which fuse the AML-1 DNA binding domain to putative transcriptional corepressors, ETO and MTG16. Here, we show that distinct domains of ETO contact the mSin3A and N-CoR corepressors and define two binding sites within ETO for each of these corepressors. In addition, of eight histone deacetylases (HDACs) tested, only the class I HDACs HDAC-1, HDAC-2, and HDAC-3 bind ETO. However, these HDACs bind ETO through different domains. We also show that the murine homologue of MTG16, ETO-2, is also a transcriptional corepressor that works through a similar but distinct mechanism. Like ETO, ETO-2 interacts with N-CoR, but ETO-2 fails to bind mSin3A. Furthermore, ETO-2 binds HDAC-1, HDAC-2, and HDAC-3 but also interacts with HDAC-6 and HDAC-8. In addition, we show that expression of AML-1-ETO causes disruption of the cell cycle in the G 1 phase. Disruption of the cell cycle required the ability of AML-1-ETO to repress transcription because a mutant of AML-1-ETO, ⌬469, which removes the majority of the corepressor binding sites, had no phenotype. Moreover, treatment of AML-1-ETO-expressing cells with trichostatin A, an HDAC inhibitor, restored cell cycle control. Thus, AML-1-ETO makes distinct contacts with multiple HDACs and an HDAC inhibitor biologically inactivates this fusion protein.The acute myeloid leukemia 1 (AML-1) gene is one of the most frequently mutated genes in human leukemia and is disrupted by multiple chromosomal translocations in AML, including t(8;21) and t(16;21) (9, 35, 38). t(8;21) is the most frequent of these translocations, and it contains the AML-1 DNA binding domain fused to a transcriptional corepressor, ETO (also known as MTG8) (4, 5, 34). t(16;21), although rarer, fuses the AML-1 DNA binding domain to an ETOrelated protein, MTG16 (9). AML-1 is also indirectly affected by inv(16), which fuses CBF␤, an allosteric regulator of AML-1, to a smooth muscle myosin heavy chain (25).ETO is highly related to MTG16 and a third family member, MTGR1, in mammalian cells and Nervy in Drosophila (6). The mammalian family members are highly conserved throughout the proteins, with four domains conserved in Nervy. These regions are an N-terminal domain that is also homologous to the transcriptional coactivator TAF110 (17), a hydrophobic heptad repeat (HHR) that mediates dimerization (3, 21), a domain of unknown function termed the Nervy domain, and a domain containing two zinc finger motifs that are required for contacting the central domain of N-CoR (29). The murine homologue of MTG16 was identified by low-stringency screening of a cDNA library by using an ETO cDNA as a probe (3). It shares 77% overall identity with human ETO, but within three of four conserved domains, these proteins are 92 to 96% identical, implying that they function similarly. The Nervy domain is the least conserved domain among family members and is 86% identical between these two proteins.ETO is a component of a high-molecular-weight complex containing ...

The accumulation of assembled holoenzymes composed of regulatory D-type cyclins and their catalytic partner, cyclin-dependent kinase 4 (cdk4), is rate limiting for progression through the G, phase of the cell cycle in mammalian fibroblasts. Both

The E2F DNA binding activity consists of a heterodimer between E2F and DP family proteins, and these interactions are required for association of E2F proteins with pRb and the pRb-related proteins p107 and p130, which modulate E2F transcriptional activities. E2F-1 expression is sufficient to release fibroblasts from G 0 and induce entry into S phase, yet it also initiates apoptosis. To investigate the mechanisms of E2F-induced apoptosis, we utilized interleukin-3 (IL-3)-dependent 32D.3 myeloid cells, a model of hematopoietic progenitor programmed cell death. In the absence of IL-3, E2F-1 alone was sufficient to induce apoptosis, and p53 levels were diminished. DP-1 alone was not sufficient to induce cell cycle progression or alter rates of death following IL-3 withdrawal. However, overexpression of both E2F-1 and DP-1 led to the rapid death of cells even in the presence of survival factors. In the presence of IL-3, levels of endogenous wild-type p53 increased in response to E2F-1, and coexpression of DP-1 further augmented p53 levels. These results provide evidence that E2F is a functional link between the tumor suppressors p53 and pRb. However, induction of p53 alone was not sufficient to trigger apoptosis, suggesting that the ability of E2F to override survival factors involves additional effectors.Members of the E2F family of transcription factors are thought to regulate cell cycle progression by activating the transcription of a set of genes necessary for the induction of S phase (30, 53). E2F DNA binding activities are dependent on growth factors (52), and their function as transcription factors is temporally regulated throughout the cell cycle by complex formation with the tumor suppressor protein pRb and the pRb-related proteins p107 and p130 (9,10,13,43,69). E2F is activated by adenovirus E1A binding to pRb and its related proteins, and release of E2F from pRb is critical for transformation induced by both E1A (18, 61) and pRb inactivation (29,31,57,58).The DNA binding activity originally termed free E2F (3) is now recognized to be a heterodimer containing the product of an E2F gene family member (E2F1 to E2F5) and a DP family member (5, 21-23, 27, 34, 38, 44, 72, 76). E2F can bind DNA in vitro, whereas DP proteins bind DNA only weakly (23). Dimerization of DP proteins with E2F proteins increases the transcriptional activity of E2F and is required for association of E2F with pRb or pRb-related proteins (4,5,21,28,41). Furthermore, enforced DP-1 expression augments E2F-mediated transformation of primary rat embryo fibroblast cells in cooperation with an activated ras oncogene (5,21,35).Microinjection of serum-starved fibroblasts with an E2F-1 expression plasmid (37) or glutathione S-transferase-E2F-1 fusion protein (17), or activation of E2F-1 expression in transfected cell lines (59,67), is sufficient to drive quiescent cells into S phase. Inappropriate entry of these cells into S phase, in the absence of survival factors, is associated with the activation

Mutations in the retinoblastoma (pRb) tumor suppressor pathway including its cyclin-cdk regulatory kinases, or cdk inhibitors, are a hallmark of most cancers and allow unrestrained E2F-1 transcription factor activity, which leads to unregulated G 1 -to-S-phase cell cycle progression. Moderate levels of E2F-1 overexpression are tolerated in interleukin 3 (IL-3)-dependent 32D.3 myeloid progenitor cells, yet this induces apoptosis when these cells are deprived of IL-3. However, when E2F activity is augmented by coexpression of its heterodimeric partner, DP-1, the effects of survival factors are abrogated. To determine whether enforced E2F-1 expression selectively sensitizes cells to cytotoxic agents, we examined the effects of chemotherapeutic agents and radiation used in cancer therapy. E2F-1 overexpression in the myeloid cells preferentially sensitized cells to apoptosis when they were treated with the topoisomerase II inhibitor etoposide. Although E2F-1 alone induces moderate levels of p53 and treatment with drugs markedly increased p53, the deleterious effects of etoposide in E2F-1-overexpressing cells were independent of p53 accumulation. Coexpression of Bcl-2 and E2F-1 in 32D.3 cells protected them from etoposide-mediated apoptosis. However, Bcl-2 also prevented apoptosis of these cells upon exposure to 5-fluorouracil and doxorubicin, which were also cytotoxic for control cells. Pretreating E2F-1-expressing cells with ICRF-193, a second topoisomerase II inhibitor that does not damage DNA, protected the cells from etoposide-induced apoptosis. However, ICRF-193 cooperated with DNA-damaging agents to induce apoptosis. Therefore, topoisomerase II inhibition and DNA damage can cooperate to selectively induce p53-independent apoptosis in cells that have unregulated E2F-1 activity resulting from mutations in the pRb pathway.Imbalance between cellular proliferation and apoptosis is a hallmark of cancer. The transcription factor E2F-1 is a critical regulator of cell cycle progression, and it plays a pivotal role in the transition from G 1 to S phase of the cell cycle (1,9,11, 22,44). The transcriptional activity of E2F-1 is negatively regulated by the product of the retinoblastoma tumor suppressor gene (pRb) (4,14,17,51) or the related family members p107 and p130 (3,6,45) and is indirectly regulated by specific cyclins, such as the D-type cyclins, their associated kinases (cdks) (23, 34, 37,45), and cdk inhibitors (p16 and p15) (15, 43). Hypophosphorylated pRb binds E2F, repressing its ability to activate genes involved in DNA synthesis and cell proliferation (e.g., dihydrofolate reductase, DNA polymerase ␣, thymidine kinase, and thymidylate synthase) (7, 35). However, when Rb is hyperphosphorylated, through the action of specific combinations of G 1 cyclins and their associated kinases (cyclin D-cdk4, cyclin D-cdk6, or cyclin E-cdk2), it releases E2F-1, which can then stimulate transcription and promote S-phase entry.Naturally occurring mutations that involve pRb have been identified in nearly every type of human ne...

The AML-1-encoded transcription factor, AML-1B, regulates numerous hematopoietic-specific genes. Inappropriate expression of AML-1-family proteins is oncogenic in cell culture systems and in mice. To understand the oncogenic functions of AML-1, we established cell lines expressing AML-1B to examine the role of AML-1 in the cell cycle. DNA content analysis and bromodeoxyuridine pulse-chase studies indicated that entry into the S phase of the cell cycle was accelerated by up to 4 h in AML-1B-expressing 32D.3 myeloid progenitor cells as compared with control cells or cells expressing E2F-1. However, AML-1B was not able to induce continued cell cycle progression in the absence of growth factors. The DNA binding and transactivation domains of AML-1B were required for altering the cell cycle. Thus, AML-1B is the first transcription factor that affects the timing of the mammalian cell cycle.The largest form of acute myeloid leukemia-1 (AML-1), 1 termed AML-1B (1) (also known as Runx1, CBFA2, or PEBP2␣B1(2-4)), activates the transcription of numerous tissue specific genes, including genes encoding cytokines and cytokine receptors, T cell receptors, and myeloid-specific genes (e.g. neutrophil peptide-3 and myeloperoxidase) (5-7). When transfected alone, AML-1B activates the transcription of these genes to low levels, but it cooperatively activates transcription to high levels in concert with tissue-specific factors (e.g. C/EBP␣, AP-1, ets-1, PU.1, and c-Myb) that regulate cellular proliferation and differentiation (7-12). Conversely, AML-1 can repress transcription by associating with the Groucho and mSin3 co-repressors (13-16).AML-1 is one of the most frequently mutated genes in human leukemia. For example, it is disrupted by the t(8;21) in AML and by the t(12;21) in childhood B-cell acute lymphoblastic leukemia (17)(18)(19). AML-1 is also targeted indirectly by the Inv (16), which fuses CBF␤, an AML-1-interacting protein, to a smooth muscle myosin heavy chain (20). However, the Inv (16) fusion protein retains the ability to interact with AML-1 and inhibits expression of AML-1 target genes (21-23). Together these chromosomal translocations account for nearly one-third of all AML and one-fourth of all childhood B-cell acute lymphoblastic leukemia cases containing discernable chromosomal abnormalities (24).Although these translocations are closely associated with acute leukemia, it is the wild-type form of AML-1 that transforms cells. Wild-type AML-1 (AML-1B) is transforming when expressed in fibroblasts, and this activity requires the C-terminal transcriptional regulatory domain (25,26). Likewise, the closely related protein PEBP2␣A1 (AML-3) is up-regulated by retroviral insertions that cooperate with c-Myc to induce T-cell lymphomas (27). Therefore, in fibroblasts and in mice, AML-1-family proteins are oncogenes. Thus, AML-1 has the unusual property that both the wild-type and the translocated alleles can affect cellular proliferation and differentiation pathways.Overexpression of the fusion protein encoded by the Inv (...

The accumulation of assembled holoenzymes composed of regulatory D-type cyclins and their catalytic partner, cyclin-dependent kinase 4 (cdk4), is rate limiting for progression through the G1 phase of the cell cycle in mammalian fibroblasts. Both the synthesis and assembly of D-type cyclins and cdk4 depend upon serum stimulation, but even when both subunits are ectopically overproduced, they do not assemble into complexes in serum-deprived cells. When coexpressed from baculoviral vectors in intact Sf9 insect cells, cdk4 assembles with D-type cyclins to form active protein kinases. In contrast, recombinant D-type cyclin and cdk4 subunits produced in insect cells or in bacteria do not assemble as efficiently into functional holoenzymes when combined in vitro but can be activated in the presence of lysates obtained from proliferating mammalian cells. Assembly of cyclin D-cdk4 complexes in coinfected Sf9 cells facilitates phosphorylation of cdk4 on threonine 172 by a cdk-activating kinase (CAK). Assembly can proceed in the absence of this modification, but cdk4 mutants which cannot be phosphorylated by CAK remain catalytically inactive. Therefore, formation of the cyclin D-cdk4 complex and phosphorylation of the bound catalytic subunit are independently regulated, and in addition to the requirement for CAK activity, serum stimulation is required to promote assembly of the complexes in mammalian cells.

Neural cell membranes naturally contain a large amount of polyunsaturated fatty acid, but the functional significance of this is unknown. An increase in membrane polyunsaturation has been shown previously to affect the high-affinity transport systems for choline and glycine in cultured human Y79 retinoblastoma cells. To test the generality of membrane polyunsaturation effects on transport, we investigated the uptake of other putative neurotransmitters and amino acids by these cells. Taurine, glutamate, and leucine were taken up by both high- and low-affinity transport systems, whereas serine, gamma-aminobutyrate, and alpha-aminoisobutyrate were taken up only by low-affinity systems. The high-affinity taurine and glutamate and low-affinity serine uptake systems were Na+ dependent. Arachidonic acid (20:4) supplementation of Y79 cells produced enrichment of all the major microsomal phosphoglycerides with 20:4, while docosahexaenoic acid (22:6) supplementation produced large increases in the 22:6 content of all fractions except the inositol phosphoglycerides. Enrichment with these polyunsaturated fatty acids facilitated taurine uptake by lowering the K'm of its high-affinity transport system. By contrast, enrichment with oleic acid did not affect taurine uptake. Glutamate, leucine, serine, gamma-aminobutyrate, and alpha-aminoisobutyrate uptake were not affected when the cells were enriched with any of these fatty acids. These findings demonstrate that only certain transport systems are sensitive to the polyunsaturated fatty acid content of the retinoblastoma cell membrane. The various transport systems either respond differently to changes in membrane lipid unsaturation, or they are located in lipid domains that are modified to different extents by changes in unsaturation.