Meropi Athanasiou

FLI-1 is a suppressor of erythroid differentiation in human hematopoietic cells.

The FLI-1 oncogene, a member of the ETS family of transcription factors, is associated with both normal and abnormal hematopoietic cell growth and lineage-specific differentiation. We have previously shown that overexpression of FLI-1 in pluripotent human hematopoietic cells leads to the induction of a megakaryocytic phenotype. In this report we show that FLI-1 also acts as an inhibitor of erythroid differentiation. Following the induction of erythroid differentiation, pluripotent cells express reduced levels of FLI-1. In contrast, when FLI-1 is overexpressed in these cells, the levels of erythroid markers are reduced. The ability of FLI-1 overexpressing cells to respond to erythroid-specific inducers such as hemin and Ara-C is also inhibited, and the uninduced cells show a reduced level of the erythroid-associated GATA-1 transcription factor mRNA. Furthermore, expression of a GATA-1 promoter-driven reporter construct in K562 cells is inhibited by co-transfection with a construct expressing FLI-1. Our results support the hypothesis that FLI-1 can act both positively and negatively in the regulation of hematopoietic cell differentiation, and that inhibition of GATA-1 expression may contribute to FLI-1-mediated inhibition of erythroid differentiation.

Cutting edge: Bone morphogenetic protein antagonists Drm/Gremlin and Dan interact with Slits and act as negative regulators of monocyte chemotaxis

Drm/Gremlin and Dan, two homologous secreted antagonists of bone morphogenic proteins, have been shown to regulate early development, tumorigenesis, and renal pathophysiology. In this study, we report that Drm and Dan physically and functionally interact with Slit1 and Slit2 proteins. Drm binding to Slits depends on its glycosylation and is not interfered with by bone morphogenic proteins. Importantly, Drm and Dan function as inhibitors for monocyte migration induced by stromal cell-derived factor 1α (SDF-1α) or fMLP. The inhibition of SDF-1α-induced monocyte chemotaxis by Dan is not due to blocking the binding of SDF-1α to its receptor. Thus, the results identify that Drm and Dan can interact with Slit proteins and act as inhibitors of monocyte chemotaxis, demonstrating a previously unidentified biological role for these proteins.

Drm/Gremlin transcriptionally activates p21Cip1 via a novel mechanism and inhibits neoplastic transformation

Drm/Gremlin, a member of the Dan family of BMP antagonists, is known to function in early embryonic development, but is also expressed in a tissue-specific fashion in adults and is significantly downregulated in transformed cells. In this report, we demonstrate that overexpression of Drm in the tumor-derived cell lines Daoy (primitive neuroectodermal) and Saos-2 (osteoblastic), either under ecdysone-inducible or constitutive promoters, significantly inhibits tumorigenesis. Furthermore, Drm overexpression in these cells increases the level of p21(Cip1) protein and reduces the level of phosphorylated p42/44 MAP kinase. Finally, our data indicate that Drm can induce p21(Cip1) transcriptionally via a novel pathway that is independent of p53 and the p38 and p42/44 MAP kinases. These results provide evidence that Drm can function as a novel transformation suppressor and suggest that this may occur through its affect on the levels of p21(Cip1) and phosphorylated p42/44 MAPK.

Role of Ets/Id proteins for telomerase regulation in human cancer cells

Most human cancers express telomerase but its activity is highly variable and regulated by complex mechanisms. Recently, we have proposed that Ets proteins may be important for regulation of telomerase activity in leukemic cells. Here we provide further evidence for the role of Ets family members and related Id proteins in telomerase regulation and characterize the underlying molecular mechanisms. By using PCR-based and gel shift assays we demonstrated specific binding to a core hTERT promoter of Ets2, Fli1, Id2, c-Myc, Mad1, and Sp1 in lysates from subclones of U937 cells. Further analysis of binding of purified proteins and various mutants of the hTERT promoter suggested the existence of a trimolecular Ets-Id2-DNA complex, and Ets inhibitory activity mediated by c-Myc and the Ets binding site on the core hTERT promoter at -293 bp from the transcription initiation site as well as a positive Ets regulatory effect mediate through another Ets binding site at -36 bp. This analysis provided evidence for the existence of negative and positive Ets regulatory site and suggested a complex interplay between Ets/Id family members and c-Myc that may be an important determinant of the diversity of telomerase activity in leukemia and other cancers.

Ets and retroviruses - transduction and activation of members of the Ets oncogene family in viral oncogenesis.

Studies of retroviral-induced oncogenesis in animal systems led to the initial discovery of viral oncogenes and their cellular homologs, and provided critical insights into their role in the neoplastic process. V-ets, the founding member of the ETS oncogene family, was originally identified as part of the fusion oncogene encoded by the avian acute leukemia virus E26 and subsequent analysis of virus induced leukemias led to the initial isolation of two other members of the ETS gene family. PU.1 was identified as a target of insertional activation in the majority of tumors induced by the murine Spleen Focus Forming virus (SFFV), while fli-1 proved to be the target of Friend murine leukemia virus (F-MuLV) in F-MuLV induced erythroleukemia, as well as that of the 10A1 and Graffi viruses. The common features of the erythroid and myeloid diseases induced by these viruses provided the initial demonstration that these and other members of the ETS family play important roles in hematopoietic development as well as disease. This review provides an overview of the role of ETS genes in retrovirally induced neoplasia, their possible mechanisms of action, and how these viral studies relate to current knowledge of the functions of these genes in hematopoiesis.

Oncogenicity of DNA in vivo: tumor induction with expression plasmids for activated H-ras and c-myc.

All vaccines and other biological products contain contaminating residual DNA derived from the production cell substrate. Whether this residual cell-substrate DNA can induce tumors in vaccine recipients and thus represent a risk factor has been debated for over 50 years without resolution. As a first step in resolving this issue, we have generated expression plasmids for the activated human H-ras oncogene and for the murine c-myc proto-oncogene. Their oncogenic activity was confirmed in vitro using the focus-formation transformation assay. Two strains of adult and newborn immune-competent mice were inoculated with different amounts of either plasmid alone or with a combination of the H-ras and c-myc plasmids. Tumors developed only in mice inoculated with both plasmids and only at the highest amount of DNA (12.5 microg of each plasmid). The NIH Swiss mouse was more sensitive than the C57BL/6 mouse, and newborn animals were more sensitive than adults. Cell lines were established from the tumors. PCR and Southern hybridization analyses demonstrated that both inoculated oncogenes were present in all of the tumor-derived cell lines and that the cells in the tumors were clonal. Western analysis demonstrated that both oncoproteins were expressed in these cell lines. These results demonstrate that cellular oncogenes can induce tumors following subcutaneous inoculation. Such information provides a possible way of evaluating and estimating the theoretical oncogenic risk posed by residual cell-substrate DNA in vaccines.

Suppression of the Ewing's sarcoma phenotype by FLI1/ERF repressor hybrids.

Fusion of the 5′ half of the Ewing’s sarcoma (ES) gene EWS with the DNA-binding domain of several transcription factors has been detected in many human tumors. The t(11;22)(q24;q12) chromosomal translocation is specifically linked to ES and primitive neuroectodermal tumors and results, in the majority of cases, in the fusion of the amino terminus of the EWS gene to the carboxyl-terminal DNA-binding domain of the FLI1 gene. The chimeric protein has been shown to be oncogenic, a potent transcriptional activator, and necessary for the maintenance of the Ewing’s phenotype, making it an attractive target for gene therapy. In this study, we demonstrate that the ES transformed phenotype can be suppressed by chimeric transcriptional repressors containing the DNA-binding domain of FLI1 and the regulatory and repressor domain of ERF, a transcription suppressor and member of the ets gene family. The hybrid repressor is expressed at levels comparable with EWS/FLI1, does not affect EWS/FLI1 expression, and exhibits similar DNA-binding specificity but suppresses transcriptional activity. The FLI1/ERF repressor, like the wild-type ERF, is regulated by mitogen-activated protein kinase-dependent subcellular localization. Our data suggest that transformation by EWS/FLI1 may partially be due to activation of specific EWS/FLI1-regulated genes involved in cell proliferation.

Cloning of the murine Drm gene (Cktsf1b1) and characterization of its oncogene suppressible promoter

The Drm gene, first identified in rat cells in our laboratory, appears to play a significant role in early embryo patterning and limb bud development. We have now isolated mouse Drm (mDrm) cDNA as well as genomic DNA clones and have mapped the Drm gene (Cktsf1b1) to murine chromosome 2. Cktsf1b1 is regulated in a tissue specific fashion and is expressed only in nontransformed mouse cells or primary fibroblasts in culture, but not in established transformed or tumor-derived mouse cell lines. The major transcription start sites map to within 69 bp upstream of the initiating ATG. A promoter was contained in the –214 to +1 bp 5′ flanking region, and promoter/reporter constructs showed 10-fold higher activity than control in REF-1 (rat), A31 (mouse) and CHO (hamster) cells. The region contains a TATA sequence and multiple potential transcription factor binding sites. Promoter activity was dose-dependently inhibited by cotransfection with either ras or mos oncogenes, but oncogene inhibition was reversed and the overall activity increased when cells were treated with the MAP kinase kinase (MKK) inhibitor PD98059. An NF-1 and Yi-like site, identified in the minimal promoter region, showed different mobility shift patterns when normal and transformed cell nuclear extracts are compared. Mutation of the NF-1 site reduced Cktsf1b1 promoter activity 25%, while mutation of the Yi-like site destroyed all the activity. Our results indicate that the expression of Cktsf1b1, a gene associated with early development and cell transformation, is sensitive to MKK levels and may be regulated via multiple transcription factor complexes.