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 t(8;21) is one of the most frequent chromosomal translocations associated with acute leukemia. This translocation creates a fusion protein consisting of the acute myeloid leukemia-1 transcription factor and the eight-twenty-one corepressor (AML1 ETO), which represses transcription through AML1 (RUNX1) DNA binding sites and immortalizes hematopoietic progenitor cells. We have identified the p14(ARF) tumor suppressor, a mediator of the p53 oncogene checkpoint, as a direct transcriptional target of AML1 ETO. AML1 ETO repressed the p14(ARF) promoter and reduced endogenous levels of p14(ARF) expression in multiple cell types. In contrast, AML1 stimulated p14(ARF) expression and induced phenotypes consistent with cellular senescence. Chromatin immunoprecipitation assays demonstrated that AML1 ETO was specifically bound to the p14(ARF) promoter. In acute myeloid leukemia samples containing the t(8;21), levels of p14(ARF) mRNA were markedly lower when compared with other acute myeloid leukemias lacking this translocation. Repression of p14(ARF) may explain why p53 is not mutated in t(8;21)-containing leukemias and suggests that p14(ARF) is an important tumor suppressor in a large number of human leukemias.

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 TELexpressing 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.The TEL (for "translocation-ETS-leukemia," also referred to as ETV6) transcription factor is a target for disruption by chromosomal translocations in several forms of acute leukemia (24-27, 38, 50, 51, 54, 57, 63). TEL was originally identified as the gene on chromosome 12 that is disrupted by t(5;12) in patients with chronic myelomonocytic leukemia (25). This translocation fuses the N-terminal homodimerization domain of TEL to the tyrosine kinase domain of the platelet-derived growth factor receptor ␤. The N terminus of TEL is also fused to the majority of the AML-1B (Runx-1) transcription factor by t(12;21), which is the most frequent translocation in pediatric B-cell acute lymphoblastic leukemias (23,26,57,61).TEL is a member of the ETS family of transcription factors. ETS factors bind heterogenous sequences centered around a core GGA sequence and cooperate with other transcription factors to regulate the transcription of a diverse set of genes (28,52,74). Several ETS factors are downstream effectors of oncogenic Ras proteins and are phosphorylated by mitogenactivated protein kinases (73,80). Aberrant expression of these ETS factors induces cellular transformation (73, 74). By contrast, TEL acts as a transcriptional repressor. In t(12;21),

No abstract

Human melanoma is a highly metastatic cancer and the regional lymph nodes are generally the first site of metastasis. Adhesion to cryostat sections of human Iymph nodes was therefore studied using two human melanoma models established from lymph node metastases, namely, MeWo cell lines of diverse metastatic potentials and a highly metastatic cell line of recent origin designated MIM/8. We found a good correlation between the metastatic potentials of the melanoma cells as measured in nude mice and their ability to adhere to cryostat sections of human lymph nodes. When adhesion to immobilized extracellular matrix proteins was measured, a significant increase in adhesion, which correlated with increased metastasis, was seen mainly on vitronectin and to a lesser extent on fibronectin. The adhesion to vitronectin and to the frozen sections were specifically blocked by an RGD-containing peptide, mAb 661 to vitronectin and mAb LM609 to integrin a,83. FACS® analysis revealed a significant and specific increase in cell surface expression of aff3 on the metastatic cells as compared to the parent line. Together these results suggest that the adhesion of melanoma cells to lymph node vitronectin via the a, 3 receptor plays a role in the process of lymphatic dissemination.

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 (...

We have reported that metastatic human melanoma cells utilize the q p 3 integrin to adhere to lymph node vitronectin (VN). In the present study, the adhesion of human and rat breast carcinoma cells to lymph node tissue was analyzed. We have previously shown a correlation between the metastatic potential of breast carcinoma cells and an RGD-mediated adhesion to cryostat sections of peripheral lymph nodes; this adhesion could be blocked by an antibody to the integrin PI subunit. Here, we show that the metastatic breast carcinoma cells were significantly more adherent to fibronectin (FN) expressed by lymph node-derived stromal cells than nonmetastatic cells. Metastatic cells also spread more rapidly than non-metastatic cells on FN-coated substrates. Using a combination of immunofluorescence microscopy, immunoprecipitation and blocking assays with integrin-specific antibodies, we found (i) that expression of the a& integrin on metastatic mammary carcinoma cells was specifically increased in comparison to non-metastatic cells and (ii) that the a$, receptor was involved in the increased adhesion of metastatic cells to lymph node FN and in cell spreading on FN-coated substrates. Our data also suggest that the integrin, which is also expressed on the metastatic cells, did not contribute to this increase in adhesion.Our data implicate the a3pI integrin in adhesion to lymph node stromal cell FN and suggest that metastatic cells of different tissue origins (e.g., melanoma and breast carcinoma) may utilize distinct integrin-ligand combinations to colonize the same target organ.o 1996 Wiley-Liss, Inc.Integrins constitute a family of heterodimers consisting of a and p subunits which mediate cell-cell and cell-substratum adhesion (Hynes, 1992). Their role in cancer progression is well documented (Albelda, 1993;Brodt, 1996). Modified expression of integrins, including up-or down-regulation of specific receptors following malignant transformation, has been described for a number of cell types (Albelda, 1993;Brodt, 1996). While these changes defy generalization and may be tumor type-and integrin-specific, there is compelling evidence to suggest that they have profound effects on the malignant phenotype (Hynes and Lander, 1992). Moreover, despite numerous reports suggesting that malignant transformation itself is associated with reduced expression of certain integrin receptors (Hynes and Lander, 1992;Albelda, 1993), the multi-step process of metastasis has repeatedly been linked to increased cell-matrix adhesion and increased expression of some matrix-binding integrin receptors (Humphries et al., 1988;Chan et al., 1991; reviewed in Brodt, 1996). This is not surprising in view of the importance of cell-matrix interactions during critical stages of metastasis, such as tumor cell arrest in the microvasculature of secondary sites, extravasation and the establishment of new secondary colonies (Zetter, 1993).The lymph node is by far the most common site of metastasis for human carcinomas (Beahrs and Myers, l983), yet relatively little i...