Biomedical subjects

S W Hiebert

Publications and source records attributed to S W Hiebert.

At least 19 recordsLinked to original sources

PubMed2006

Histone deacetylase inhibitors induce the degradation of the t(8;21) fusion oncoprotein.

The t(8;21) chromosomal translocation that generates the fusion oncoprotein RUNX1-ETO predominates in leukemia patients of the French-American-British (FAB) class M2 subtype. The oncoprotein has the capacity to promote expansion of hematopoietic stem/progenitor cells and induces leukemia in association with other genetic alterations. Here, we show that RUNX1-ETO undergoes degradation in response to treatment with histone deacetylase inhibitors, one of which, depsipeptide (DEP), is currently undergoing phase II clinical testing in a variety of malignancies. These compounds induce turnover of RUNX1-ETO without affecting the stability of RUNX1-ETO partner proteins. In addition, RUNX1-ETO physically interacts with heat shock protein 90 (HSP90). DEP treatment interrupts the association of RUNX1-ETO with HSP90 and induces proteasomal degradation of RUNX1-ETO. DEP and the HSP90 antagonist 17-allylamino-geldanamycin (17-AAG) both triggered RUNX1-ETO degradation, but without any additive or cooperative effects. These findings may stimulate the development of more rational and effective approaches for treating t(8;21) patients using histone deacetylase inhibitors or HSP90 inhibitors.

Cell Line, Tumor↗

PubMed2006

RUNX1 associates with histone deacetylases and SUV39H1 to repress transcription.

RUNX1 (AML1) is a gene that is frequently disrupted by chromosomal translocations in acute leukemia. Like its Drosophila homolog Runt, RUNX1 both activates and represses transcription. Both Runt and RUNX1 are required for gene silencing during development and a central domain of RUNX1, termed repression domain 2 (RD2), was defined as being required for transcriptional repression and for the silencing of CD4 during T-cell maturation in thymic organ cultures. Although transcriptional co-repressors are known to contact other repression domains in RUNX1, the factors that bind to RD2 had not been defined. Therefore, we tested whether RD2 contacts histone-modifying enzymes that may mediate both repression and gene silencing. We found that RD2 contacts SUV39H1, a histone methyltransferase, via two motifs and that endogenous Suv39h1 associates with a Runx1-regulated repression element in murine erythroleukemia cells. In addition, one of these SUV39H1-binding motifs is also sufficient for binding to histone deacetylases 1 and 3, and both of these domains are required for full RUNX1-mediated transcriptional repression. The association between RUNX1, histone deacetylases and SUV39H1 provides a molecular mechanism for repression and possibly gene silencing mediated by RUNX1.

Animals↗

PubMed2004

Modern diagnostics in chronic myeloproliferative diseases (CMPDs).

According to the new WHO classification a group of chronic myeloproliferative diseases (CMPDs) were defined: chronic myeloid leukemia (CML), chronic neutrophilic leukemia (CNL), chronic eosinophilic leukemia and hypereosinophilic syndrome (CEL/HES), polycythemia vera (PV), chronic idiopathic myelofibrosis (with extramedullary hematopoiesis, CIMF), essential thrombocythemia (ET), and so called CMPD/unclassifiable. As clinical features and laboratory findings differ widely between these diseases several diagnostic approaches are mandatory at diagnosis for classification and are needed also for follow up studies, especially for the measurement of minimal residual disease (MRD). We here outline the laboratory set up at diagnosis and during follow up in CMPDs with specific focus on the respective therapeutical consequences. Only by using a comprehensive diagnostic panel including cytomorphology, cytogenetics, and molecular genetic methods establishing the correct diagnosis, optimizing treatment as well as evaluating treatment response is possible in CMPDs today.

Blood Cell Count↗

PubMed2001

Bcl-2 is an apoptotic target suppressed by both c-Myc and E2F-1.

Malignant transformation occurs in cells that overexpress c-Myc or that inappropriately activate E2F-1. Transformation occurs after the selection of cells that have acquired resistance to apoptosis that is triggered by these oncogenes, and a key mediator of this cell death process is the p53 tumor suppressor. In IL-3-dependent immortal 32D.3 myeloid cells the ARF/p53 apoptotic pathway is inactivated, as these cells fail to express ARF. Nonetheless, both c-Myc and E2F-1 overexpression accelerated apoptosis when these cells were deprived of IL-3. Here we report that c-Myc or E2F-1 overexpression suppresses Bcl-2 protein and RNA levels, and that restoration of Bcl-2 protein effectively blocks the accelerated apoptosis that occurs when c-Myc- or E2F-1-overexpressing cells are deprived of IL-3. Blocking p53 activity with mutant p53 did not abrogate E2F-1-induced suppression of Bcl-2. Analysis of immortal myeloid cells engineered to overexpress c-Myc and E2F-1 DNA binding mutants revealed that DNA binding activity of these oncoproteins is required to suppress Bcl-2 expression. These results suggest that the targeting of Bcl-2 family members is an important mechanism of oncogene-induced apoptosis, and that this occurs independent of the ARF/p53 pathway.

Animals↗

PubMed2001

TEL contacts multiple co-repressors and specifically associates with histone deacetylase-3.

TEL (Translocation-ETS-Leukemia or ETV 6) is disrupted by multiple chromosomal translocations in acute leukemia. The loss of heterozygosity at the TEL locus in leukemias and the hemizygous deletion of TEL that is observed in various tumors, suggests that TEL is a tumor suppressor. Overexpression of TEL alters cellular morphology and represses the expression of the matrix metalloproteinase stromelysin-1. Based on these studies, deletion analysis was used to define the minimal repression domains of TEL. TEL-mediated repression required both the N-terminal pointed domain and a central region composed of amino acids 268-303. The mSin3A and N-CoR corepressors bind to the pointed domain and the central repression domain of TEL, respectively. Unexpectedly, histone deacetylase-3, but not other histone deacetylases, also associates with the central region of TEL. Histone deacetylase-3 interacts with a TEL mutant that cannot bind N-CoR, suggesting that this is a direct interaction with TEL. In addition, histone H3 was under-acetylated near the TEL-binding sites in the endogenous stromelysin-1 promoter when TEL was expressed. Furthermore, trichostatin A, a potent histone deacetylase inhibitor, impaired TEL-dependent repression of the stromelysin-1 promoter. Finally, while TEL-expression induced cellular aggregation of Ras-transformed cells, Trichostatin A reversed the TEL-induced cellular aggregation phenotype. Thus, the cumulative data suggests that histone deacetylase-3 activity is required for the transcriptional functions of TEL.

3T3 Cells↗

PubMed2001

E2F-1 induces the stabilization of p53 but blocks p53-mediated transactivation.

E2F-1 induces p53 accumulation and E2F-1 and p53 form a physical complex, which affects the ability of E2F-1 to activate transcription. We mapped the domains on E2F-1 that interact with p53 and found two p53-binding domains. To understand the functional consequences of the E2F-1/p53 association on p53 activities we identified the domains of E2F-1 that were responsible for the accumulation of p53. Unexpectedly, we found that the E2F-1 transactivation domain was dispensable for p53 induction. By contrast, further deletion of the DP-1 interaction/'marked' box domain eliminated p53 accumulation. Radiolabeling pulse/chase analysis demonstrated that E2F-1 caused post-translational stabilization of p53. Although E2F-1 caused the stabilization of p53, E2F-1 expression impaired p53-dependent transactivation. Thus, the E2F-1 : p53 interaction may provide a checkpoint function to inactivate overactive E2F-1, but the association may also inactivate p53 transactivation to allow cell cycle progression.

Animals↗

PubMed2001

Role of co-repressors in transcriptional repression mediated by the t(8;21), t(16;21), t(12;21), and inv(16) fusion proteins.

The t(8;21), t(16;21), inv(16), and t(12;21) are some of the most frequent chromosomal translocations found in acute myeloid and acute lymphoblastic leukemia. The fusion proteins created by these chromosomal translocations are transcriptional repressors. A full understanding of the types of proteins that these fusion proteins recruit to repress transcription will not only clarify understanding of the molecular mechanism of action of these fusion proteins but also provide further targets for therapeutic intervention.

Acute Disease↗

PubMed2001

ETO, a target of t(8;21) in acute leukemia, makes distinct contacts with multiple histone deacetylases and binds mSin3A through its oligomerization domain.

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, Delta469, 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.

Amino Acid Sequence↗

PubMed2000

AML-1/ETO fusion protein is a dominant negative inhibitor of transcriptional repression by the promyelocytic leukemia zinc finger protein.

The AML-1/ETO fusion protein, created by the (8;21) translocation in M2-type acute myelogenous leukemia (AML), is a dominant repressive form of AML-1. This effect is due to the ability of the ETO portion of the protein to recruit co-repressors to promoters of AML-1 target genes. The t(11;17)(q21;q23)-associated acute promyelocytic leukemia creates the promyelocytic leukemia zinc finger PLZFt/RAR alpha fusion protein and, in a similar manner, inhibits RAR alpha target gene expression and myeloid differentiation. PLZF is expressed in hematopoietic progenitors and functions as a growth suppressor by repressing cyclin A2 and other targets. ETO is a corepressor for PLZF and potentiates transcriptional repression by linking PLZF to a histone deacetylase-containing complex. In transiently transfected cells and in a cell line derived from a patient with t(8;21) leukemia, PLZF and AML-1/ETO formed a tight complex. In transient assays, AML-1/ETO blocked transcriptional repression by PLZF, even at substoichiometric levels relative to PLZF. This effect was dependent on the presence of the ETO zinc finger domain, which recruits corepressors, and could not be rescued by overexpression of co-repressors that normally enhance PLZF repression. AML-1/ETO also excluded PLZF from the nuclear matrix and reduced its ability to bind to its cognate DNA-binding site. Finally, ETO interacted with PLZF/RAR alpha and enhanced its ability to repress through the RARE. These data show a link in the transcriptional pathways of M2 and M3 leukemia. (Blood. 2000;96:3939-3947)

Binding Sites↗

PubMed2000

Role of the transcription factor AML-1 in acute leukemia and hematopoietic differentiation.

Chromosomal translocations affecting the AML-1 gene are among the most frequent aberrations found in acute leukemia. Because the AML-1 transcription factor is a critical regulator of hematopoeitic cell development, normal homeostasis is disrupted in cells containing these translocations. In this review we describe the mechanisms of transcriptional activation and repression by AML-1 and how this transcriptional control is disrupted by the chromosomal translocations that affect AML-1. Finally, we discuss how the mechanism of transcriptional repression by these chromosomal translocation fusion proteins is a possible target of therapeutic intervention in acute leukemia.

Acute Disease↗

PubMed2000

Expression of the AML-1 oncogene shortens the G(1) phase of the cell cycle.

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.

Animals↗

PubMed2000

A mechanism of repression by acute myeloid leukemia-1, the target of multiple chromosomal translocations in acute leukemia.

AML1 is one of the most frequently translocated genes in human leukemia. Here we demonstrate that acute myeloid leukemia-1 (AML-1) (Runx-1) represses transcription from a native promoter, p21(Waf1/Cip1). Unexpectedly, this repression did not require interactions with the Groucho co-repressor. To define the mechanism of repression, we asked whether other co-repressors could interact with AML-1. We demonstrate that AML-1 interacts with the mSin3 co-repressors. Moreover, endogenous AML-1 associated with endogenous mSin3A in mammalian cells. A deletion mutant of AML-1 that did not interact with mSin3A failed to repress transcription. The AML-1/mSin3 association suggests a mechanism of repression for the chromosomal translocation fusion proteins that disrupt AML-1.

Acute Disease↗

PubMed2000

Alterations in subnuclear trafficking of nuclear regulatory factors in acute leukemia.

The nuclear matrix plays an important role in the functional organization of the nucleus in part by locally concentrating regulatory factors involved in nucleic acid metabolism. A number of nuclear regulatory proteins initially identified due to their involvement in human cancer are localized to discrete nuclear matrix-attached foci and correct nuclear partitioning likely plays a role in their function. Two such examples are promyelocytic leukemia (PML) and acute myelogenous leukemia-1 (AML-1; Runx1). PML, the target of the t(15;17) in acute PML, is localized to PML nuclear bodies (also termed Nuclear Domain 10 and PML oncogenic domains), a nuclear matrix-associated body whose function appears to be quite complex, with probable roles in cancer, apoptosis, and in acute viral infections. In t(15;17)-containing leukemic cells, the PML nuclear bodies are disrupted, but reform when the leukemic cells are induced to differentiate in the presence of all-trans retinoic acid. AML1 (RUNX1) is a key regulator of hematopoietic differentiation and AML1 proteins are found in nuclear compartments that reflect their roles in transcriptional activation and repression. The t(8;21), associated with AML, results in a chimeric transcription factor, AML-1/ETO (eight twenty one), that remains attached to the nuclear matrix through targeting signals contained in the ETO protein. When co-expressed, ETO and AML-1/ETO co-localize to a nuclear compartment distinct from that of AML1 or PML nuclear bodies. Interestingly, enforced expression of ETO or AML-1/ETO changes the average number of PML nuclear bodies per cell. Thus, chromosomal translocations involving AML1 result in altered nuclear trafficking of the transcription factor as well as other changes to the nuclear architecture. J. Cell. Biochem. Suppl. 35:93-98, 2000.

Animals↗

PubMed2000

TEL, a putative tumor suppressor, modulates cell growth and cell morphology of ras-transformed cells while repressing the transcription of stromelysin-1.

TEL is a member of the ETS family of transcription factors that interacts with the mSin3 and SMRT corepressors to regulate transcription. TEL is biallelically disrupted in acute leukemia, and loss of heterozygosity at the TEL locus has been observed in various cancers. Here we show that expression of TEL in Ras-transformed NIH 3T3 cells inhibits cell growth in soft agar and in normal cultures. Unexpectedly, cells expressing both Ras and TEL grew as aggregates. To begin to explain the morphology of Ras-plus TEL-expressing cells, we demonstrated that the endogenous matrix metalloproteinase stromelysin-1 was repressed by TEL. TEL bound sequences in the stromelysin-1 promoter and repressed the promoter in transient-expression assays, suggesting that it is a direct target for TEL-mediated regulation. Mutants of TEL that removed a binding site for the mSin3A corepressor but retained the ETS domain failed to repress stromelysin-1. When BB-94, a matrix metalloproteinase inhibitor, was added to the culture medium of Ras-expressing cells, it caused a cell aggregation phenotype similar to that caused by TEL expression. In addition, TEL inhibited the invasiveness of Ras-transformed cells in vitro and in vivo. Our results suggest that TEL acts as a tumor suppressor, in part, by transcriptional repression of stromelysin-1.

3T3 Cells↗

PubMed2000

The ETO protein disrupted in t(8;21)-associated acute myeloid leukemia is a corepressor for the promyelocytic leukemia zinc finger protein.

The ETO protein was originally identified by its fusion to the AML-1 transcription factor in translocation (8;21) associated with the M2 form of acute myeloid leukemia (AML). The resulting AML-1-ETO fusion is an aberrant transcriptional regulator due to the ability of ETO, which does not bind DNA itself, to recruit the transcriptional corepressors N-CoR, SMRT, and Sin3A and histone deacetylases. The promyelocytic leukemia zinc finger (PLZF) protein is a sequence-specific DNA-binding transcriptional factor fused to retinoic acid receptor alpha in acute promyelocytic leukemia associated with the (11;17)(q23;q21) translocation. PLZF also mediates transcriptional repression through the actions of corepressors and histone deacetylases. We found that ETO is one of the corepressors recruited by PLZF. The PLZF and ETO proteins associate in vivo and in vitro, and ETO can potentiate transcriptional repression by PLZF. The N-terminal portion of ETO forms complexes with PLZF, while the C-terminal region, which was shown to bind to N-CoR and SMRT, is required for the ability of ETO to augment transcriptional repression by PLZF. The second repression domain (RD2) of PLZF, not the POZ/BTB domain, is necessary to bind to ETO. Corepression by ETO was completely abrogated by histone deacetylase inhibitors. This identifies ETO as a cofactor for a sequence-specific transcription factor and indicates that, like other corepressors, it functions through the action of histone deactylase.

Acute Disease↗

PubMed2000

Topoisomerase IIalpha mediates E2F-1-induced chemosensitivity and is a target for p53-mediated transcriptional repression.

Mutations of the retinoblastoma tumor suppressor, pRb, or its cyclin-cyclin-dependent kinase (CDK) regulatory kinases or CDK inhibitors, allows unrestrained E2F activity, leading to unregulated cell cycle progression. However, overexpression of E2F-1 also sensitizes cells to apoptosis, suggesting that targeting this pathway may be of therapeutic benefit. Enforced expression of E2F-1 in interleukin-3-dependent myeloid cells led to preferential sensitivity to the topoisomerase II inhibitor, etoposide, which was independent of p53 accumulation. Pretreatment of the E2F-1-expressing cells with ICRF-193, a second topoisomerase II inhibitor that does not cause DNA damage, protected these cells against etoposide-induced apoptosis. However, ICRF-193 cooperated with other DNA-damaging agents to induce apoptosis. Enforced expression of E2F-1 led to accumulation of p53 protein. An E2F-1 mutant that is defective in inducing cell cycle progression also induced p53, suggesting that p53 was responding directly to E2F, and not to secondary events caused by inappropriate cell cycle progression (i.e., DNA damage). Thus, topoisomerase II inhibition and DNA damage cooperate to selectively induce apoptosis in cells that have mutations in the pRb pathway.

Antigens, Neoplasm↗

PubMed1999

The inv(16) encodes an acute myeloid leukemia 1 transcriptional corepressor.

The inv(16) is one of the most frequent chromosomal translocations associated with acute myeloid leukemia (AML). The inv(16) fusion protein acts by dominantly interfering with AML-1/core binding factor beta-dependent transcriptional regulation. Here we demonstrate that the inv(16) fusion protein cooperates with AML-1B to repress transcription. This cooperativity requires the ability of the translocation fusion protein to bind to AML-1B. Mutational analysis and cell fractionation experiments indicated that the inv(16) fusion protein acts in the nucleus and that repression occurs when the complex is bound to DNA. We also found that the inv(16) fusion protein binds to AML-1B when it is associated with the mSin3A corepressor. An AML-1B mutant that fails to bind mSin3A was impaired in cooperative repression, suggesting that the inv(16) fusion protein acts through mSin3 and possibly other corepressors. Finally, we demonstrate that the C-terminal portion of the inv(16) fusion protein contains a repression domain, suggesting a molecular mechanism for AML-1-mediated repression.

Animals↗

PubMed1999

Identification and characterization of a protein containing formin homology (FH1/FH2) domains.

A novel member of the Formin/Diaphanous family of proteins was cloned and characterized. A 4kB mRNA is ubiquitously expressed but is found in abundance in the spleen. FHOS (Formin Homologue Overexpressed in Spleen) contains a 3414bp open reading frame and encodes for an approximately 128kDa protein. FHOS has sequence homology to Diaphanous and Formin proteins within the Formin Homology (FH)1 and FH2 domains. FHOS also contains a coiled-coil, a collagen-like domain, two nuclear localization signals, and several potential PKC and PKA phosphorylation sites. FHOS-specific antiserum was generated and used to determine that FHOS is a predominantly cytoplasmic protein and is expressed in a variety of human cell lines. FHOS was mapped to chromosome 16q22 between framework markers WI-5594 and WI-9392.

Amino Acid Sequence↗