Oncogene database

2113

ETS1

v-ets avian erythroblastosis virus E26 oncogene homolog 1;ETS1;v-ets avian erythroblastosis virus E26 oncogene homolog 1

Avian erythroblastosis virus E26 (v-ets) oncogene homolog-1|protein C-ets-1|v-ets avian erythroblastosis virus E2 oncogene homolog 1

11q23.3

protein-coding

The adenomatous polyposis coli (APC) tumor suppressor protein is inactivated by mutations in the majority of colorectal cancers. A recent study has revealed that alterations in the APC signaling pathway can result in the transcriptional activation of the c-MYC gene.

Glycoprotein (GP) IX is a subunit of the von Willebrand receptor, GPIb-V-IX, which mediates adhesion of platelets to the subendothelium of damaged blood vessels. Previous characterization of the GPIX promoter identified a functional Ets site that, when disrupted, reduced promoter activity. However, the Ets protein(s) that regulated GPIX promoter expression was unknown. In this study, transient cotransfection of several GPIX promoter/reporter constructs into 293T kidney fibroblasts with a Fli-1 expression vector shows that the oncogenic protein Fli-1 can transactivate the GPIX promoter when an intact GPIX Ets site is present. In addition, Fli-1 binding of the GPIX Ets site was identified in antibody supershift experiments in nuclear extracts derived from hematopoietic human erythroleukemia cells. Comparative studies showed that Fli-1 was also able to transactivate the GPIbalpha and, to a lesser extent, the GPIIb promoter. Immunoblot analysis identified Fli-1 protein in lysates derived from platelets. In addition, expression of Fli-1 was identified immunohistochemically in megakaryocytes derived from CD34(+) cells treated with the megakaryocyte differentiation and proliferation factor, thrombopoietin. These results suggest that Fli-1 is likely to regulate lineage-specific genes during megakaryocytopoiesis.

The protein encoded by the Ets-1 proto-oncogene is a transcription factor that regulates expression of matrix proteases. It has been associated with tumor invasion and angiogenesis. Glioma progression is characterized by increased invasiveness and neovascularization, so we hypothesized that expression of Ets-1 proto-oncogene might play a role in the progression of these tumors. Therefore, we examined the expression of Ets-1 protein by immunohistochemical means and in situ hybridization in tissues obtained from 81 primary and 20 recurrent astrocytic tumors. Twenty-eight (65%) of 43 glioblastomas (Grade IV astrocytomas) stained for Ets-1. The percentage of positive cells in glioblastomas varied from 10 to 90%. Of the 16 anaplastic astrocytomas (Grade III), 4 (25%) were moderately positive (<50% of cells) for Ets-1. None of 22 cases of low-grade astrocytomas (Grade II) expressed endogenous Ets-1. The staining score was significantly associated with tumor grade (P < .0001). Normal brain tissues did not express Ets-1 protein, whereas recurrent astrocytoma cases expressed significantly more positivity for Ets-1 than did primary tumors (P = .03). The Ets-1 protein was observed mainly in the nucleus and corresponded to the cytoplasmic Ets-1 mRNA localization by in situ hybridization. Western and Northern blot analyses confirmed overexpression of Ets-1 protein and mRNA in high-grade tumors. We conclude that Ets-1 protein expression correlates with the malignant potential of tumors of astroglial origin.

We have analyzed gene expression in hemopoietic and testicular cell types after their exposure to 50 MHz radiofrequency (RF) non-ionizing radiation modulated (80%) with a 16 Hz frequency. The exposure system generates a 0.2 microT magnetic field parallel to the ground and a 60 V/m electric field orthogonal to the earth s magnetic field. Exposure conditions were selected so as to interfere with the calcium ion flow. Under these electromagnetic field (EMF) conditions, we observed an overexpression of the ets1 mRNA in Jurkat T-lymphoblastoid and Leydig TM3 cell lines. This effect was observed only in the presence of the 16 Hz modulation, corresponding to the resonance frequency for calcium ion with a DC magnetic field of 45.7 microT. We have also identified a putative candidate gene repressed after EMF exposure. The experimental model described in this paper may contribute to the understanding of the biological mechanisms involved in EMF effects.

The v-ets oncoprotein and its progenitor Ets1 belong to a family of transcription factors that are related by an 85 amino acid conserved DNA binding domain, the ets domain. Ets1 plays important role(s) in control of cell proliferation, differentiation and apoptosis. Abnormal expression of Ets1 could lead to disruption of these processes and contribute to development of malignancy. Retinoic acid (RA) inhibits proliferation, induces differentiation and regulates apoptosis in many different cell types. Here, we demonstrate that RA treatment increases the expression of Ets1 mRNA, but not that of Ets2, Elk1 or Fli1 in MC3T3-E1 cells. Ets1 induction is detectable after 4 h, can be maintained for at least 14 days, and is inhibited by Actinomycin D, which suggests that RA regulation of Ets1 occurs at the transcriptional level. The promoter region of Ets1 contains four retinoic acid response element (RARE) half sites located at -94, -152, -1765 and -2252 from the translation start site. We show that RARbeta is expressed by MC3T3-E1 cells in the presence of RA and demonstrate that it binds to the -94 RARE half site. Furthermore, RA induces transcription of Ets1 promoter-reporter constructs containing this RARE half site.

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.

Elk-1, a c-Fos protooncogene regulator, which belongs to the ETS-domain family of transcriptional factors, plays an important role in the induction of immediate early gene expression in response to a variety of extracellular signals. In this study, we demonstrate for the first time the in vitro and in vivo interaction of Elk-1 with BRCA1 splice variants BRCA1a and BRCA1b using GST-pull down assays, co-imunoprecipitations/Western blot analysis of cell extracts from breast cancer cells and mammalian two-hybrid assays. We have localized the BRCA1 interaction domain of Elk-1 protein to the conserved ETS domain, a motif involved in DNA binding and protein-protein interactions. We also observed binding of BRCA1 proteins to other ETS-domain transcription factors SAP1, ETS-1, ERG-2 and Fli-1 but not to Elk-1 splice variant DeltaElk-1 and c-Fos protooncogene. Both BRCA1a and BRCA1b splice variants function as growth suppressors of human breast cancer cells. Interestingly, our studies reveal that although both Elk-1 and SAP-1 are highly homologous members of a subfamily of ETS domain proteins called ternary complex factors, it is only Elk-1 but not SAP-1 that can augment the growth suppressive function of BRCA1a/1b proteins in breast cancer cells. Thus Elk-1 could be a potential downstream target of BRCA1 in its growth control pathway. Furthermore, we have observed inhibition of c-Fos promoter activity in BRCA1a transfected stable breast cancer cells and over expression of BRCA1a/1b attenuates MEK-induced SRE activation in vivo. These results demonstrate for the first time a link between the growth suppressive function of BRCA1a/1b proteins and signal transduction pathway involving Elk-1 protein. ALL these results taken together suggest that one of the mechanisms by which BRCA1a/1b proteins function as growth/tumor suppressors is through inhibition of the expression of Elk-1 target genes like c-Fos.

The proto-oncogene, ets-1, is a transcription factor known to control the expression of a number of genes involved in extracellular matrix remodeling and has been postulated to play a role in cell migration and tumor invasion. To elucidate the involvement of ets-1 in human colorectal carcinomas, we examined 41 cases of colorectal adenoma and 122 cases of colorectal carcinoma by immunohistochemistry and compared the degree of Ets-1 expression with the depth of carcinoma invasion. In adenomas, 12 of 41 cases (29.3%) showed immuno-positivity for Ets-1. 12 of 27 cases (44.4%) of adenoma with high grade dysplasia showed immunopositivity for Ets-1. However, there was no positive case in low or moderate dysplasia of adenoma. In contrast, 103 of 122 cases (84.4%) of colorectal adenocarcinoma showed immunoreactivity for Ets-1 in the carcinoma cells themselves. We investigated the relationship between pathological features in colorectal carcinoma and Ets-1 immunoreactivity of the tumor cells. Among the 122 cases of invasive carcinomas, Ets-1 immunoreactivity was significantly correlated with the depth grading of tumor invasion (P < .0001), the presence of lymph node metastasis (P < .05), lymphatic invasion (P < .01) and venous invasion (P < .05). However, Ets-1 expression did not correlate with histological differentiation. In situ hybridization also confirmed the presence of ets-1 mRNA in colorectal carcinomas. expression of ets-1 mRNA was also detected in two of three human colorectal carcinoma tissues and in four of six different kinds of carcinoma cell lines by the reverse transcription polymerase chain reaction method. These findings suggest that the expression of Ets-1 is one of the important factors related to carcinogenesis and/or tumor invasion of colorectal carcinoma.

mutations in the c-KIT receptor occur somatically in many sporadic Gastrointestinal Stromal tumors (GIST), and similar mutations have been identified at the germline level in kindreds with multiple GISTs. These mutations activate the tyrosine kinase activity of c-KIT and induce constitutive signaling. To investigate the function of activated c-KIT in GIST, we established a human GIST cell line, GIST882, which expresses an activating KIT mutation (K642E) in the first part of the cytoplasmic split tyrosine kinase domain. Notably, the K642E substitution is encoded by a homozygous exon 13 missense mutation, and, therefore, GIST882 cells do not express native KIT. GIST882 c-KIT protein is constitutively tyrosine phosphorylated, but tyrosine phosphorylation was rapidly and completely abolished after incubating the cells with the selective tyrosine kinase inhibitor STI571. Furthermore, GIST882 cells evidenced decreased proliferation and the onset of apoptotic cell death after prolonged incubation with STI571. Similar results were obtained after administering STI571 to a primary GIST cell culture that expressed a c-KIT exon 11 juxtamembrane mutation (K558NP). These cell-culture-based studies support an important role for c-KIT signaling in GIST and suggest therapeutic potential for STI571 in patients afflicted by this chemoresistant tumor.

BACKGROUND/AIMS: Ets-1 regulates the expression of a number of genes related to remodeling of the extracellular matrix. Ets-1 is associated with the occurrence of invasive processes, proliferation and differentiation. Less is known about the biological functions of Ets-1 in human hepatocellular carcinoma. In an attempt at elucidation, we examined immunohistochemically hepatocellular carcinoma followed by application of genetic techniques. METHODOLOGY: We performed immunohistochemical analysis on tissue from 59 Japanese patients undergoing surgical resection of hepatocellular carcinoma using the antibody against human Ets-1. We compared Ets-1 expression in relation to clinicopathological findings. In situ hybridization and reverse transcription-polymerase chain reaction were also performed to confirm the expression of Ets-1 messenger RNA in hepatocellular carcinoma tissues. RESULTS: In specimens from 59 patients with hepatocellular carcinoma, 41 (70%) showed positive staining for Ets-1 protein. The expression of Ets-1 messenger RNA was also observed in hepatocellular carcinoma tissues by in situ hybridization and reverse transcription-polymerase chain reaction. The expression of Ets-1 correlated with histological differentiation of hepatocellular carcinoma (P < 0.05). Ets-1 was positive in 2 (50%) of the 4 well-differentiated hepatocellular carcinomas and in 28 (64%) of the 44 moderately differentiated hepatocellular carcinomas, whereas ALL the 11 poorly differentiated hepatocellular carcinomas were positive for Ets-1 staining. Ets-1 protein was expressed more strongly at the peripheral than the central area of the tumor. Otherwise, no particular correlation was evident in terms of clinicopathological factors. CONCLUSIONS: We found Ets-1 to be expressed in human hepatocellular carcinoma, particularly at the peripheral area of the tumor. As this expression is linked to cell differentiation, this gene may yield biological information relative to this malignant tumor of the liver.

The chromosomal translocation specifically linked to the Ewing sarcoma family results in the generation of fusion proteins comprising the amino terminal portion of EWS and the DNA-binding domain of ets transcription factors. The EWS/ets chimeric proteins act as aberrant transcription factors leading to tumorigenic processes. We searched for genes specifically activated in Ewing sarcoma cells but not in other tumor cell lines using the gene array technique, and found significantly enhanced expression of the Id2 gene. High levels of Id2 transcripts were detected in Ewing sarcoma cell lines and tumor tissues. The EWS/ets chimeric proteins activated the Id2 gene via the 5 -upstream promoter sequence. Chromatin-immunoprecipitation revealed a direct interaction of EWS/Fli-1 with the promoter regions of the Id2, TGF-beta type II receptor, cyclin D1, and c-myc genes. Since EWS/Fli-1 transactivates c-myc, a cooperative action of the chimeric protein and c-myc leads to overexpression of Id2. In the present study, we suggest that Id2 is a target of the chimeric proteins and that the c-myc/Id2 pathway plays a pivotal role in the tumorigenic processes provoked by EWS/ets proteins.

Despite significant structural diversity, present evidence suggests that EWS/ETS fusion proteins promote oncogenesis by transcriptionally modulating a common set of target genes. In order to identify these genes, microarray expression analyses were performed on NIH 3T3 polyclonal populations expressing one of three EWS/ETS fusion genes. The majority of these genes can be grouped into seven functional categories, including cellular metabolism and signal transduction. The biologic significance of these target genes was pursued. The effects of modulating genes involved in metabolism were assessed by flux studies and demonstrated shifts in glucose utilization and lactate production as a result of EWS/FLI1 expression. The proto-oncogene coding for serine/threonine kinase PIM3 was found to one of several genes encoding signal transduction proteins that were up-regulated by EWS/ETS fusions. PIM3 was found to be expressed in a panel of human Ewing s family tumor cell lines. Forced expression of PIM3 promoted anchorage-independent growth. Coexpression of a kinase-deficient PIM3 mutant attenuated EWS/FLI1-mediated NIH 3T3 tumorigenesis in immunodeficent mice.

Ets-1 oncoprotein is a transcription factor known to regulate the expression of numerous genes important in extracellular matrix remodeling and angiogenesis. Up-regulation of Ets-1 has been shown to be important in a variety of human malignancies and to correlate with prognosis. To our knowledge, this oncoprotein has not been examined in melanocytic lesions. A series of 10 cutaneous melanomas and 24 benign melanocytic lesions with patient records were independently examined for diagnosis confirmation and immunohistochemical expression by two dermatopathologists. The immunohistochemical expression for Ets-1 (Novocastra, Newcastle upon Tyne, UK) was scored by an average of the mean labeling intensity; no nuclear staining = 0, weak nuclear staining = 1, moderate = 2, and intense = 3. Ets-1 expression was statistically assessed by the one-way analysis of variance (ANOVA) comparing the mean labeling intensity of melanoma to benign melanocytic nevi. ALL of the benign melanocytic lesions exhibited negative to weak nuclear staining, with an average mean labeling intensity of 0.4. Melanoma in situ exhibited moderate nuclear staining, for a mean labeling intensity of 2.0, whereas ALL conventional invasive melanomas exhibited moderate to strong nuclear staining, with a mean labeling intensity of 2.7. Metastatic melanoma exhibited very strong nuclear staining, with a mean labeling intensity of 3.0. Invasive desmoplastic melanoma, like melanoma in situ, showed moderate nuclear staining with a mean labeling intensity of 2.1. There was a trend toward more intense staining with melanoma progression. A statistically significant difference in the mean labeling intensity of Ets-1 was seen between invasive melanoma and benign melanocytic nevi (P <.0001). Ets-1 oncoprotein expression, however, does not distinguish among benign melanocytic lesions. Staining intensity and pattern might be a useful adjunct with histomorphology in distinguishing invasive melanoma from benign melanocytic nevi. Furthermore, Ets-1 expression may be an important pathogenic mechanism and predictor of aggressive biologic behavior of cutaneous melanoma, with a trend toward staining intensity increasing as Clark stage increases.

The Ets1 proto-oncoprotein is a member of the Ets family of transcription factors that share a unique DNA binding domain, the Ets domain. The DNA binding activity of Ets1 is controlled by kinases and transcription factors. Some transcription factors, such as AML-1, regulate Ets1 by targeting its autoinhibitory module. Others, such as Pax-5, alter Ets1 DNA binding properties. Ets1 harbors two phosphorylation sites, threonine-38 and an array of serines within the exon VII domain. Phosphorylation of threonine-38 by ERK1/2 activates Ets1, whereas phosphorylation of the exon VII domain by CaMKII or MLCK inhibits Ets1 DNA binding activity. Ets1 is expressed by numerous cell types. In haemotopoietic cells, it contributes to the regulation of cellular differentiation. In a variety of other cells, including endothelial cells, vascular smooth muscle cells and epithelial cancer cells, Ets1 promotes invasive behavior. Regulation of MMP1, MMP3, MMP9 and uPA as well as of VEGF and VEGF receptor gene expression has been ascribed to Ets1. In tumors, Ets1 expression is indicative of poorer prognosis.

The influence of a polymorphic variant of the ETS-1 oncogene on the predisposition to develop chronic B-cell leukemia (CLL) was investigated. A total of 59 patients with CLL and 59 controls were examined for the frequency of an XbaI restriction fragment length polymorphism RFLP of the ETS-1 oncogene which has been reported to occur more frequently in patients with hematological malignancies than in normal controls. We found no significant difference in the allele frequency between the CLL patients and the normal controls. These data suggest that the presence of the XbaI RFLP is not associated with CLL.

The c-ets1 proteins are transcriptional activators expressed within endothelial cells during blood vessel development in chick embryos. The authors show by in situ hybridization that c-ets1 is transcribed in the endothelia during angiogenesis in human embryos, in granulation tissue, and especially during tumor vascularization. c-ets1 mRNAs were also detected in the fibrocytes of tumor stroma and in the spindle cells of Kaposi s sarcomas, regarded as cells of endothelial origin. It has been shown that the c-ets proteins activate transcription through a PEA3 motif that plays a role in the stimulation of transcription of urokinase-type plasminogen-activator (u-PA), stromelysin and collagenase genes. The authors demonstrate in vitro that the angiogenic factor TNF alpha increases transiently the amount of both c-ets1 and u-PA mRNA in confluent human umbilical vein endothelial cells. Therefore, the authors suggest that the c-ets1 proteins might regulate the transcription of the genes coding for matrix-degrading proteases, which are necessary for both angiogenesis and tumor invasion.

The beta factor, which interacts with the rpL32 promoter, binds to the sequence 5 -GAGCCGGAAGTG and trans-activates this gene. Comparison of the DNA sequences bound by the beta factor with those bound by other known DNA-binding proteins revealed that the ETS proteins interact with similar DNA sequences. Consequently we have examined the relationship of the beta factor to the several ETS proteins so far reported. Antibody and oligonucleotide competition experiments, performed by using electrophoretic shift analysis, revealed that the beta factor contains ETS epitopes and that it is immunologically related to both of the GA-binding proteins (GABPs), implying that the beta factor may consist of two separate protein subunits.

The capacity of glioma cells to invade normal brain leads to far reaching dissemination of these tumors limiting surgical resection and the prospect of local treatment strategies. The analysis of the dissemination pattern, the molecular substrates and mechanisms involved suggest that glioma invasion most likely represents independent cellular behaviors leading to distinct pattern of spread. The search for common denominators of the invasive phenotype has demonstrated that invasive cells show gene expression profiles indicating elevated expression of motility associated genes and genes involved in resistance to apoptosis whereas proliferation and apoptosis related gene expression is repressed. Confirming these findings, invasive cells in vitro show elevated motility and resistance to drug induced apoptosis. Obviously invasiveness is an early event in the progression of glial tumors. Therefore, the loss of control over invasion genes must involve key elements of molecular tumor progression. We have recently demonstrated that deregulation of invasion gene expression occurs as a consequence of functional impairment of tumor suppressor p53 leading to unrestrained activation of proto-oncogene Ets-1 dependent invasion-associated genes. In this article the prospect of limiting the dissemination of glial tumors by anti-invasive strategies and targets for modulation of the invasive phenotype to reconstitute chemo- and radiation sensitivity are discussed.

cancer formation and progression is a complex process determined by several mechanisms that promote cell growth, invasiveness, neo-angiogenesis, and render neoplastic cells resistant to apoptosis. The tumor suppressor p53 and the proto-oncogenic factor ets-1 are important regulators of such mechanisms. While it is well established that p53 and ets-1 influence various aspects of cell behavior by regulating the transcription of specific genes, little is known about the functional relationship between these transcription factors. We found that the gene encoding thromboxane synthase (TXSA), which we recently identified as a factor promoting invasion and resistance to apoptosis in gliomas, is a novel target gene for both p53 and ets-1. We demonstrate that p53 and ets-1 coregulate TXSA in an antagonistic and inter-related manner, with ets-1 being a potent transcriptional activator and p53 inhibiting ets-1-dependent transcription. Negative interference with ets-1 transcription requires functional p53 and is lost in mutant p53 proteins. We show that ets-1 and p53 associate physically in vitro and in vivo and that their interaction, rather than a direct binding of p53 to the TXSA promoter, is required for transcriptional repression of TXSA by wild-type p53. An important implication of our findings is that the loss of p53-mediated negative control over ets-1-dependent transcription may lead to the acquisition of an invasive phenotype in tumor cells.

We have continued the molecular analysis of D-elg, a member of the Drosophila ets gene family. Based on the characterization of cDNA and genomic sequences, the D-elg gene contains five exons and four introns and produces a mRNA with an open reading frame of 464 amino acids. Consistent with this analysis, in vitro translation of a near full-length D-elg cRNA yields a protein with a molecular weight of approximately 56 kDa. D-elg shows significant homology to other ets proteins in the amino-terminal A domain and strong homology in the carboxy-terminal ETS domain. The D-elg protein is most similar to the alpha-subunit of the mouse GA-binding protein.

Interferon (IFN) regulatory factor-1 (IRF-1) is a transcription factor that has been historically associated with type I IFN activation and antioncogenic properties. We studied IRF-1 expression and DNA-binding capacity in nontransformed and transformed mouse fibroblasts. A 43-kDa nuclear IRF-1 protein was expressed biphasically during the cell cycle in primary mouse embryo fibroblasts, nontransformed NIH 3T3 cells, and ras revertants. IRF-1 expression became constitutive in ras-transformed NIH 3T3 cells and in cells transformed by oncogenes ets, fes, fos, her-2/neu, met, mos, raf, or trk, suggesting that deregulated IRF-1 expression may be associated with loss of growth control. Lysyl oxidase (LO), a ras suppressor that is downregulated in ras transformants, is an IRF-1 target gene, but it is not stimulated by abundant IRF-1 present in transformants, while another IRF-1 target gene (iNOS) is transcribed. IRF-1 from either normal or ras-transformed cells bound to IRF elements in the IFN-beta and LO promoters. IRF-1 in transformants can, therefore, bind to but not transactivate the LO promoter, and the presence of IRF-1 is not sufficient to suppress ras transformation. LO expression may effect the regulated expression of IRF-1: a ras revertant, which was generated by stable transfection of LO cDNA, regained the normal biphasic IRF-1 pattern. A mainly cytoplasmic, constitutively expressed 46-kDa protein with immunologic identity to the 43-kDa nuclear IRF-1 was also present in normal and transformed cells, but as it did not bind to the IRF elements, its function is unclear.

The c-ets1 gene product (Ets1) is the prototype of a family of sequence-specific transcriptional activators which have been implicated in various developmental processes and in the response of cells to a variety of extracellular stimuli. We report here a structure-function analysis of the DNA binding and transcriptional activation properties of Ets1. The minimal region required for specific DNA binding is located at the carboxy-terminus of Ets1, a domain highly conserved in ALL known members of the Ets family. Transcriptional activation by Ets1 in mammalian cells requires an additional domain of 110 amino acids characterized by a high content of acidic residues and localized in the amino-terminal half of the protein. This domain also functions as a transcriptional activation domain in yeast cells when linked to the heterologous DNA binding domain of Gal4. In contrast to its conservation in Ets1 proteins across vertebrate species, this activation domain is not conserved in other members of the Ets family. These results indicate that an important level of specificity between different members of the Ets family may reside in the differential interactions of their respective activation domains with distinct general transcription factors or different associated coactivators.

The human ETS1 proto-oncogene proteins have been isolated from the T-cell leukemia line, CEM, by immunoaffinity chromatography and their identity confirmed by NH2-terminal amino acid sequencing. Incubation of CEM cells with N alpha-p-tosyl-L-lysine chloromethyl ketone (TLCK) indicates that ETS proteins can be modified in their cellular context and that pretreatment of the cells with N-ethylmaleimide (NEM) protects ETS1 proteins from TLCK modification. These data show that ETS1 proteins can exist in at least two different states, -SH-available and -SH-protected. Renatured human ETS1 has DNA sequence-specific binding to the PEA3 (CAGGAAGT) motif. The ETS1.PEA3 complex can be observed by electrophoretic mobility shift assays (EMSA). Purified ETS1 retards a band which is exactly the same size as a complex that is retarded from nuclear extracts prepared from CEM cells. Reduced ETS1 is required to form the ETS1.PEA3 complex, however; modification of the ETS1 -SH groups by either NEM or by TLCk does not inhibit formation of the complex. The ETS1.PEA3 complex formed with TLCK-modified ETS1 has a slower mobility than the complex formed with unmodified ETS1. Zone sedimentation analysis of purified ETS1 indicates that it is the monomer of ETS1 which binds to the PEA3 oligonucleotide.

BACKGROUND: Ets-1 is a transactivator of matrix-associated genes and key factor in neoangiogenesis. The expression of Ets-1 mRNA and protein was analyzed in healthy rat kidney and in a model for mesangioproliferative disease without and with inhibition of platelet-derived growth factor-B (PDGF-B) activity. METHODS: Immunohistochemistry was performed using a specific noncrossreacting anti-Ets-1 antibody and included a counterstain with alpha-smooth muscle actin (alpha-SMA). Nuclear proteins from isolated glomeruli were analyzed by Western blotting. Changes in Ets-1 mRNA levels were detected by in situ hybridization and Northern blotting. PDGF-B antagonism was performed in nephritic rats by specific aptamers. RESULTS: The distribution of Ets-1-positive cells in healthy rats was heterogenous with exclusively nuclear staining of glomerular, tubular, and vascular cells. Profound changes were detected in the anti-Thy 1.1 glomerulonephritis. Nuclear Ets-1 staining was intense in mesangial cells, whereas podocyte and endothelial cell staining was unchanged. The strongest signal was seen on days 2 to 7. By Western blotting of glomerular proteins a single 52 kD band was detected in healthy rats, which was increased 4.5-fold after disease induction. At the same time a 54 kD band appeared that most likely represents phosphorylated Ets-1. Ets-1 transcripts were detected in mesangial cells that include exon IV but lack exon VII. A concordant 6.4-fold up-regulation of mRNA was detected in glomeruli. Specific PDGF-B antagonism by aptamer treatment from days 3 to 7 after disease induction led to reduced Ets-1 expression on day 7, correlating with decreased mesangial cell numbers. CONCLUSION: A temporal increase of mesangial cell Ets-1 expression correlates with mesangial cell activation in mesangioproliferative glomerulonephritis. PDGF-B may partially contribute to the increased expression.

Ets proteins are transcription factors, which share a unique DNA binding domain, the Ets domain. Some members of the Ets family are implicated in tumorigenesis. Ets1, the founder of the Ets family, is predominantly expressed in invasive tumors and able to activate certain genes encoding ECM-degrading proteases. We used RNA-interference in combination with DNA chip analysis to identify Ets1-regulated genes in MDA-MB-231 breast cancer cells. Of the Ets1-responsive proteases, matrix metalloproteases MMP1 and MMP9, but not MMP3 or uPA, showed reduced RNA levels when endogenous Ets1 expression was suppressed. These data suggest that Ets1 regulates only a certain subset of ECM-degrading proteases. How Ets1 is regulated in invasive breast cancer cells is unknown. The observations that protein kinase C inhibitors abrogated Ets1 expression and that protein kinase C was able to increase Ets1-dependent transcription imply that protein kinase C is a potential regulator of Ets1 activity in breast cancer cells.

E1AF is an ets-oncogene family transcription factor. E1AF was shown to upregulate multiple matrix metalloproteinase (MMP) genes and contribute to the malignant phenotype of cancer cells by inducing invasive and metastatic activities. E1AF is upregulated by hepatocyte growth factor (HGF) stimulation, which indicates that E1AF would participate in cell motility by HGF/scatter factor. On the other hand, E1AF upregulates p21waf1/cip1 to induce cell cycle arrest when cells are exposed to stress. EWS/ETS fusions are frequently observed in Ewing s sarcoma, and we have revealed that EWS/ETS chimeric protein activates telomerase activity by upregulating hTERT. However, substitution ets binding site (EBS) mutants did not affect the responsiveness to EWS/E1AF. DNA-IP assay showed that the complexes contained EWS/E1AF bound to the hTERT promoter, which suggested that EWS/ETS functions as a co-activator for TERT transcription. Our findings that EWS/ETS acts as a transcriptional co-factor may imply that the transcription pathway is regulated by the interaction of transcription factors.

The proto-oncogene, ets-1, is a transcription factor known to control the expression of a number of genes and has been postulated to play a role in cell growth, differentiation and tumour invasion. We examined 137 cases of breast carcinoma by immunohistochemistry and compared the degree of Ets-1 expression among the different histological types of invasive carcinomas. Ets-1 was not expressed in the normal breast epithelium nor in noninvasive carcinomas. Among the 137 breast carcinoma cases, 104 (83.2%) showed positive staining for the Ets-1 protein. Histologically, invasive ductal carcinomas expressed immunopositivity with intense staining for Ets-1 in the tumour cells. Ets-1 expression correlated with Bloom-Richardson grading in invasive ductal carcinoma (p<0.01). However, there was no correlation between Ets-1 expression and lymph node metastasis, "t" classification or TNM staging. In situ hybridization confirmed the presence of Ets-1 mRNA in breast carcinomas. The expression of Ets-1 mRNA was detected in two of three different kinds of cultured human breast carcinoma cell lines and one of three human breast carcinoma tissues by the reverse transcription polymerase chain reaction method. These findings suggest that ets-1 is overexpressed in ductal cells of the breast that have undergone malignant conversion and that ets-1 is one of the factors associated with tumour growth and histological differentiation of breast carcinomas.

AIMS: The high incidence of clinically diagnosed prostatic cancer is exceeded by the frequency of tumours detected at autopsy. The Ets-1 proto-oncogene is expressed by a variety of malignant and normal tissues. Therefore, in this study, expression of Ets-1 protein was investigated in latent prostatic cancer detected at autopsy, compared with benign prostatic hyperplasia, normal prostatic tissues and clinical prostatic cancer. METHODS AND RESULTS: Using immunohistochemistry, we analysed Ets-1 expression in 95 prostatic specimens including 19 cases of latent prostatic carcinoma (LPC) and 55 cases of clinical prostatic carcinoma (CPC), 11 cases of benign prostatic hyperplasia (BPH) and 10 cases of normal prostate (NP). Differences in the incidence of LPC and CPC suggest different courses for the biological progression of prostatic cancer. There was a significant difference in the degree of Ets-1 expression in CPC and LPC (P < 0.05). Ets-1 was not expressed in BPH and NP, but in malignant cases (57 of 74; 77.0%) commonly demonstrated immunoreactivity in the tumour cells. In our study the expression of Ets-1 between benign and malignant, and well, moderately and poorly differentiated adenocarcinomas of prostatic cancer showed significant differences. The presence of Ets-1 mRNA was confirmed by in-situ hybridization in human prostatic tissues. CONCLUSION: Our results suggest that Ets-1 might play an important role in carcinogenesis and/or the progression of human prostatic carcinomas.

Histamine is known to act, at least in part, as a growth factor for several cell types, and as production of this biogen amine has been found to accelerate the rate of tissue proliferation in wound repair, embryogenesis and malignant growth. Abundant experimental and clinical data suggest that histamine augments in vivo tumour cell proliferation via histamine H2 receptors (H2R). Here, we report that exogenously added histamine stimulates Ets-1 (v-ets erythroblastosis virus E26 oncogene homolog 1) synthesis in human melanoma cells. Involvement of histamine receptors in the histamine induced ets-1 expression has been also studied. Our data show that these newly recognized actions of histamine are mediated by the H2R. Modification of local protooncogen Ets-1 level is likely being involved in the regulation of melanoma growth.

BACKGROUND: Ets-1 proto-oncogene exhibits multiple activities in the transcriptional regulation of numerous genes including metalloproteinase (MMP)-1, -3 and -9. MMPs play an important role in the remodelling of extracellular matrix in various renal diseases. However, the role of the Ets-1-MMP axis in advanced renal diseases is uncertain. In the present study, we investigated whether Ets-1 is involved in interleukin (IL)-1-mediated expression of MMPs in tubulointerstitial cells. METHODS: Rat renal fibroblasts (NRK-49F) and tubular epithelial cells (NRK-52E) were cultured and allocated to an IL-1beta-treated group (10 ng/ml), a platelet-derived growth factor (PDGF)-BB-treated group (25 ng/ml) and a control group. Protein and mRNA were extracted after 1, 6, 12 and 24 h of treatment. Parallel flasks were treated with 2 muM ets-1 antisense oligodeoxynucleotides (ODNs) before exposure to IL-1beta. The expression of Ets-1 protein was evaluated by western blotting. The activities of MMPs were evaluated by gelatin zymography. The expression of ets-1 and/or MMP-9 mRNA was evaluated semiquantitatively by real-time reverse transcription-polymerase chain reaction (RT-PCR). RESULTS: In NRK-49F cells, Ets-1 protein increased significantly by 6.8-fold at 6 h, and MMP-9 activity increased significantly by 9.9-fold at 12 h in the IL-1beta-treated group compared with controls. MMP-2 and -3 activities also increased significantly in the IL-1beta-treated group. In NRK-52E cells, Ets-1 protein was 3.1 times higher at 1 h, and the latent form of MMP-9 activity increased 3.4-fold at 6 h in the IL-1beta group compared with controls. However, MMP-2 or MMP-3 activities were not markedly altered by IL-1beta treatment compared with controls. When the cells were treated with ets-1 antisense ODNs before IL-1beta treatment, Ets-1 protein expression decreased at least 50%, and MMP-9 activity was clearly inhibited in both cells. We also confirmed that MMP-9 activity was upregulated on days 21 and 28 in renal cortex of rat crescentic glomerulonephritis. CONCLUSIONS: The Ets-1 transcriptional factor may participate in IL-1beta-mediated MMP-9 expression in tubulointerstitial cells.

TEL2/ETV7 is highly homologous to the ETS transcription factor TEL/ETV6, a frequent target of chromosome translocation in human leukemia. Although both proteins are transcriptional inhibitors binding similar DNA recognition sequences, they have opposite biologic effects: TEL inhibits proliferation while TEL2 promotes it. In addition, forced expression of TEL2 but not TEL blocks vitamin D3-induced differentiation of U937 and HL60 myeloid cells. TEL2 is expressed in the hematopoietic system, and its expression is up-regulated in bone marrow samples of some patients with leukemia, suggesting a role in oncogenesis. Recently we also showed that TEL2 cooperates with Myc in B lymphomagenesis in mice. Here we show that forced expression of TEL2 alone in mouse bone marrow causes a myeloproliferative disease with a long latency period but with high penetrance. This suggested that secondary mutations are necessary for disease development. Treating mice receiving transplants with TEL2-expressing bone marrow with the chemical carcinogen N-ethyl-N-nitrosourea (ENU) resulted in significantly accelerated disease onset. Although the mice developed a GFP-positive myeloid disease with 30% of the mice showing elevated white blood counts, they ALL died of T-cell lymphoma, which was GFP negative. Together our data identify TEL2 as a bona fide oncogene, but leukemic transformation is dependent on secondary mutations.

AIM: Gastrointestinal stromal tumors (GISTs) are rare. GISTs differ from other mesenchymal tumors of the gastrointestinal tract (e.g. leiomyomas and schwannomas). The purpose of this study was to investigate the role of Ets-1 in the growth and differentiation of GISTs. METHODS: Twenty-eight GISTs, nine leiomyomas and six schwannomas were examined by immunohistochemical staining method for Ets-1 in this study. Specimens were selected from surgical pathology archival tissues at Nagasaki University Hospital. RESULTS: Ets-1 protein was expressed in the cytoplasm of cells in ALL of these tumors. Immunohistochemical staining revealed that 27 GISTs (96.4%), six leiomyomas (66.7%), and five schwannomas (83.3%) were positive for Ets-1. Ets-1 expression was statistically different between GISTs and leiomyomas (P<0.005). However, there was no correlation between Ets-1 expression and clinical risk categories. CONCLUSION: Ets-1 plays an important role in the growth and differentiation of GISTs, leiomyomas and schwannomas.

BACKGROUND: The transcription factor avian erythroblastosis virus E26 (V-Ets) oncogene homolog 1 (Ets-1) is involved in tumor development and progression through the transcriptional regulation of several matrix-degrading enzyme systems, including matrix metalloproteinases (MMPs). It has been demonstrated that the MMPs are expressed strongly in high-grade meningiomas. To determine the biologic significance of Ets-1 in the progression of benign meningiomas, the authors investigated the expressions of Ets-1 and its target genes MMP-2 and MMP-9 in primary and recurrent, Grade 1 meningiomas. METHODS: The expression levels of Ets-1, MMP-2, and MMP-9 were examined by immunohistochemistry in 70 Grade 1 meningiomas, including 36 primary tumors without recurrence after 5 years of follow-up and 17 pairs of primary tumors and subsequent recurrences. RESULTS: The results demonstrated higher expression of Ets-1, MMP-2, and MMP-9 proteins in meningiomas with subsequent recurrences compared with meningiomas from patients who had no recurrences (P < .001). In addition, Ets-1 expression was correlated with the expression of both MMP-2 and MMP-9. CONCLUSIONS: Ets-1 may be involved in meningioma recurrence by up-regulating MMP-2 and MMP-9. Increased expression of these genes in World Health Organization grade 1 meningiomas may serve as an indicator for a high risk of recurrence.

Translocations fusing the strong androgen-responsive gene, TMPRSS2, with ERG or other oncogenic ETS factors may facilitate prostate cancer development. Here, we studied 18 advanced prostate cancers for ETS factor alterations, using reverse transcription-PCR and DNA and RNA array technologies, and identified putative ERG downstream gene targets from the microarray data of 410 prostate samples. Out of the 27 ETS factors, ERG was most frequently overexpressed. Seven cases showed TMPRSS2:ERG gene fusions, whereas the TMPRSS2:ETV4 fusion was seen in one case. In five out of six tumors with high ERG expression, array-CGH analysis revealed interstitial 2.8 Mb deletions between the TMPRSS2 and ERG loci, or smaller, unbalanced rearrangements. In silico analysis of the ERG gene coexpression patterns revealed an association with high expression of the histone deacetylase 1 gene, and low expression of its target genes. Furthermore, we observed increased expression of WNT-associated pathways and down-regulation of tumor necrosis factor and cell death pathways. In summary, our data indicate that the TMPRSS2:ERG translocation is common in advanced prostate cancer and occurs by virtue of unbalanced genomic rearrangements. Activation of ERG by fusion with TMPRSS2 may lead to epigenetic reprogramming, WNT signaling, and down-regulation of cell death pathways, implicating ERG in several hallmarks of cancer with potential therapeutic importance.

Infection by Kaposi s sarcoma-associated herpesvirus (KSHV) is required for the development of Kaposi s sarcoma (KS), a highly inflammatory angiogenic tumor of endothelial cells commonly found in untreated AIDS patients. Angiopoietin 2 (Ang-2) modulates the vasculature during inflammation and angiogenesis, but the mechanism by which KSHV regulates Ang-2 expression has not been investigated. Here, we show that KSHV infection of primary human umbilical vein endothelial cells induced the expression and release of Ang-2, which in turn was required for KSHV-induced paracrine-dependent angiogenesis in vivo. Ang-2 was strongly expressed in small vessels and spindle tumor cells in KS tumors. Mechanistically, KSHV activated the Ang-2 promoter via AP-1 and Ets1 transcriptional factors, which were mediated by ERK, JNK, and p38 mitogen-activated protein kinase (MAPK) pathways. Our findings demonstrate the importance of Ang-2 in KS angiogenesis and define a novel role for AP-1 and MAPK pathways in regulating angiogenesis. This study also illustrates a distinct mechanism by which a tumor virus modulates vasculature to promote tumorigenesis and exemplifies the convergence of oncogenesis and angiogenesis pathways in tumor development.

The ets oncogene superfamily consists of a family of sequence-specific DNA-binding proteins that activate transcription. We have previously identified two new members of the ets oncogene superfamily, namely elk-1 and elk-2. In this report we show that the recombinant elk-1 protein expressed in bacteria, like the c-ets-1 proto-oncogene, binds in a sequence-specific manner to Moloney murine sarcoma virus long terminal repeat, E74 target sequences and the PEA3 motif (polyoma enhancer), but does not bind to PU box sequences. Thus analysis of the DNA-binding specificity of ets-related proteins supports the view that different members show similar DNA-binding specificity, which is a general feature of the homeobox proteins. Our data using the chloramphenicol acetyltransferase gene linked to a thymidine kinase promoter containing multimers of the elk-1 target sequence indicates that elk-1 functions as a transcriptional activator. Interestingly, although elk-1 is the most divergent of ALL the members of the ets gene family, it shows very close similarities with c-ets-1 in some of its sequence-specific DNA-binding specificities. Here, we propose a new function for the elk-1 gene to act as a transcriptional activator of retroviruses and DNA tumor viruses.

Recently, we identified recurrent gene fusions involving the 5 untranslated region of the androgen-regulated gene TMPRSS2 and the ETS (E26 transformation-specific) family genes ERG, ETV1 or ETV4 in most prostate cancers. Whereas TMPRSS2-ERG fusions are predominant, fewer TMPRSS2-ETV1 cases have been identified than expected on the basis of the frequency of high (outlier) expression of ETV1 (refs 3-13). Here we explore the mechanism of ETV1 outlier expression in human prostate tumours and prostate cancer cell lines. We identified previously unknown 5 fusion partners in prostate tumours with ETV1 outlier expression, including untranslated regions from a prostate-specific androgen-induced gene (SLC45A3) and an endogenous retroviral element (HERV-K_22q11.23), a prostate-specific androgen-repressed gene (C15orf21), and a strongly expressed housekeeping gene (HNRPA2B1). To study aberrant activation of ETV1, we identified two prostate cancer cell lines, LNCaP and MDA-PCa 2B, that had ETV1 outlier expression. Through distinct mechanisms, the entire ETV1 locus (7p21) is rearranged to a 1.5-megabase prostate-specific region at 14q13.3-14q21.1 in both LNCaP cells (cryptic insertion) and MDA-PCa 2B cells (balanced translocation). Because the common factor of these rearrangements is aberrant ETV1 overexpression, we recapitulated this event in vitro and in vivo, demonstrating that ETV1 overexpression in benign prostate cells and in the mouse prostate confers neoplastic phenotypes. Identification of distinct classes of ETS gene rearrangements demonstrates that dormant oncogenes can be activated in prostate cancer by juxtaposition to tissue-specific or ubiquitously active genomic loci. Subversion of active genomic regulatory elements may serve as a more generalized mechanism for carcinoma development. Furthermore, the identification of androgen-repressed and insensitive 5 fusion partners may have implications for the anti-androgen treatment of advanced prostate cancer.

The proto-oncogene Ets-1 is a transcription factor that is known to regulate certain matrix metalloproteinases and plasminogen activator, which have been associated with malignant behaviors in solid carcinomas. We hypothesized that Ets-1 expression is also associated with tumor progression and a worse prognosis in lung carcinoma patients. To clarify the role of the Ets-1 proto-oncogene, the expression of Ets-1 in non-small cell lung carcinomas using 156 paraffin-embedded specimens was determined in surgically resected tissue samples. Immunohistochemical staining showed Ets-1 expression in 82 cases of 156 carcinomas (53%): 36 of 52 (69%) squamous cell carcinomas, 41 of 96 (43%) adenocarcinomas, and 5 of 8 (63%) other carcinomas. In adenocarcinomas, a higher proportion of acinar type expressed Ets-1 compared to papillary or alveolar type (p < 0.05). The proportion of adenocarcinoma that expressed Ets-1 increased with poorer histologic differentiation of the adenocarcinoma (p < 0.05). Ets-1 positive adenocarcinomas had a larger mean size than Ets-1 negative adenocarcinomas (p < 0.01). In adenocarcinoma patients, expression of Ets-1 was associated with disease-free (p = 0.09) and overall survivals (p < 0.05) after lung resection. Such relationship was not observed among squamous cell carcinoma patients. Our findings indicate that Ets-1 expression is related to histopathological differentiation, morphogenesis, and tumor progression of lung adenocarcinomas. Ets-1 appears to be a useful predictor of poor prognosis after surgical resection in lung adenocarcinoma patients. Ets-1 expression could be used to evaluate the malignant behaviors of lung adenocarcinomas.

There is evidence that specificity proteins (Sp), such as Sp1, Sp3, and Sp4, are overexpressed in tumors and contribute to the proliferative and angiogenic phenotype associated with cancer cells. Sp1, Sp3, and Sp4 are expressed in a panel of estrogen receptor (ER)-positive and ER-negative breast cancer cell lines, and we hypothesized that regulation of their expression may be due to microRNA-27a (miR-27a), which is also expressed in these cell lines and has been reported to regulate the zinc finger ZBTB10 gene, a putative Sp repressor. Transfection of ER-negative MDA-MB-231 breast cancer cells with antisense miR-27a (as-miR-27a) resulted in increased expression of ZBTB10 mRNA and decreased expression of Sp1, Sp3, and Sp4 at the mRNA and protein levels and also decreased activity in cells transfected with constructs containing Sp1 and Sp3 promoter inserts. In addition, these responses were accompanied by decreased expression of Sp-dependent survival and angiogenic genes, including survivin, vascular endothelial growth factor (VEGF), and VEGF receptor 1 (VEGFR1). Moreover, similar results were observed in MDA-MB-231 cells transfected with ZBTB10 expression plasmid. Both as-miR-27a and ZBTB10 overexpression decreased the percentage of MDA-MB-231 cells in S phase of the cell cycle; however, ZBTB10 increased the percentage of cells in G(0)-G(1), whereas as-miR-27a increased the percentage in G(2)-M. This latter response was associated with induction of Myt-1 (another miR-27a target gene), which inhibits G(2)-M through enhanced phosphorylation and inactivation of cdc2. Thus, the oncogenic activity of miR-27a in MDA-MB-231 cells is due, in part, to suppression of ZBTB10 and Myt-1.

The c-ets protooncogenes have recently been shown to code for transcription factors that activate the oncogene responsive unit of the polyoma virus enhancer. We show that transcription of the stromelysin gene, which is highly expressed in transformed cells and tumours, is efficiently activated by c-Ets-1 and -2 through two DNA elements. The distal element is a highly conserved palindrome composed of two strong binding sites for c-Ets-1. The proximal element does not bind c-Ets-1, but may be activated indirectly by increased synthesis of c-Jun and c-Fos. Both ets responsive elements mediate activation by the oncoproteins Ha-Ras, v-Src and v-Mos. These results suggest that c-Ets participates in the mechanisms by which stromelysin gene expression is deregulated in transformed cells and tumours.

The successful treatment of breast cancer is dependent upon a number of complex factors. Her-2/neu gene amplification is known to be one of the most common genetic alterations associated with breast cancer and its accurate determination has become necessary for the selection of patients for trastuzumab therapy. The aim of this study was to prove the consistency of chromogenic in situ hybridisation (CISH) technique after analyzing the overexpression of the Her-2/neu proto-oncogene in 100 invasive breast carcinomas and by comparing CISH results with immunohistochemistry (IHC) and fluorescence in situ hybridisation (FISH). Moreover, it was done to evaluate the possible correlation of estrogen (ERs) and progesterone receptors (PRs), the proliferation marker Ki67 and the tumour suppressor gene p53 with HER-2/neu status of these breast carcinomas. Of the 100 breast carcinomas that were analysed, 22 cases showed HER-2/neu amplification, 66 cases showed no amplification, whereas 12 cases were non-interpretable in both assays (FISH and CISH). Consequently, the overall concordance between FISH and CISH was 100%. Additionally, it was observed that when HER-2/neu gene was overexpressed, there was an association with negative PRs and ERs status, negative p53 protein expression and high Ki67 labelling index. It is concluded that patients with tumours scoring 2+ with the CBE356 antibody (borderline immunohistochemistry-tested cases) would also benefit from CISH as it is shown to be highly accurate, practical and can be easily integrated into routine testing in any histopathology laboratory. Finally, CISH represents an important addition to the HER2 testing algorithm.

Recent studies have demonstrated that the nuclear protein, Ets-1, which is preferentially expressed in lymphocytes, binds to the long terminal repeat of Moloney murine sarcoma virus and HTLV-1 and regulates gene expression. The association of Ets-1 with DNA has been shown to be lost when the protein is phosphorylated. Thus, Ets-1 may regulate gene expression in lymphocytes and this activity may be determined by its phosphorylation state. To address the possibility that Ets-1 activity may be altered by membrane (m) Ig-mediated signal transduction, we analyzed the effect of mIgM and mIgD ligation on the phosphorylation state of Ets-1. Monoclonal anti-IgM or anti-IgD antibody stimulation of normal mouse B cells led to increased phosphorylation of Ets-1 within 2 min. This response was absolutely dependent on calcium mobilization and could be induced by elevation of intracellular free calcium using the calcium ionophore, ionomycin. Calcium release from intracellular stores was sufficient to mediate the phosphorylation of Ets-1. Treatment of resting B cells with IL-4, TGF beta-1, IFN-gamma, anti-class I, or anti-class II antibodies did not induce Ets-1 phosphorylation. In summary, calcium mobilization from intracellular stores after mIgM or mIgD ligation provides a necessary and sufficient signal for activation of Ets-1 phosphorylation. This phosphorylation event may act in the alteration of gene expression during B cell activation.

We studied a novel restriction fragment length polymorphism (RFLP) of the proto-oncogenes ETS-1 that we detected in a patient with an acute monocytic leukemia by the presence of two, 3.7 and 10 kb, Xbal fragments on Southern blots of DNA from blast cells and remission blood samples. RFLP analysis of a series of 114 normal donors revealed that only four (3.6%) shared the 10 kb fragment. By contrast, this unusual allele was found in 20 (all lymphocytic or monocytic) of 108 (18.5%) hematological malignancies (p less than 0.001). DNA sequence analysis indicated the disappearance in the rare allele of a Xbal site due to a single point mutation at the 3 end of ETS-1 locus. Molecular consequences of this mutation point to a possible pathogenic involvement of ETS-1 in these disorders and to the question of genetic susceptibility to hematological malignancies.

BACKGROUND: Ets-1 is a widely expressed transcription factor implicated in several biological processes including hematopoiesis, where it contributes to the regulation of cellular differentiation. The functions of Ets-1 are regulated by transcription factors as well as by phosphorylation events: phosphorylation of threonine 38 activates Ets-1, whereas phosphorylation of a cluster of serines within exon VII reduces DNA binding activity. This study focuses on the role of Ets-1 during granulocytic differentiation of NB4 promyelocytic and HL60 myeloblastic leukemia cell lines induced by all-trans retinoic acid. DESIGN AND METHODS: Ets-1 expression was measured by real-time reverse transcriptase polymerase chain reaction and western blotting. The role of Ets-1 during all-trans retinoic acid-induced differentiation was analyzed by using a transdominant negative molecule or small interfering RNA. RESULTS: NB4 and HL60 cell lines expressed high levels of p51 Ets-1, while the splice variant isoform that lacks exon VII (p42) was almost undetectable. The addition of all-trans retinoic acid reduced p51 Ets-1 levels and induced inhibitory phosphorylation of the remaining protein. expression of Ets-1 was also reduced during dimethylsulfoxide-induced differentiation and during granulocytic differentiation of human CD34(+) hematopoietic progenitor cells but not in NB4.R2 and HL60R cells resistant to all-trans retinoic acid. In line with these observations, transduction of a transdominant negative molecule of Ets-1, which inhibited DNA binding and transcriptional activity of the wild-type Ets-1, significantly increased chemical-induced differentiation. Consistently, Ets-1 knockdown by small interfering RNA increased the number of mature neutrophils upon addition of all-trans retinoic acid. Interestingly, p51 Ets-1 over-expression was frequently observed in CD34(+) hematopoietic progenitor cells derived from patients with acute myeloid leukemia, as compared to its expression in normal CD34(+) cells. CONCLUSIONS: Our results indicated that a decreased expression of Ets-1 protein generalizes to granulocytic differentiation and may represent a crucial event for granulocytic maturation.

The molecular mechanisms of glomerulosclerosis and tubulointerstitial fibrosis in diabetic nephropathy (DN) have received scant attention. Ets-1 proto-oncogene plays a role in matrix remodeling by regulating matrix-degrading enzymes. We investigated the possible role of Ets-1 in the pathogenesis of DN. 6-week-old male Sprague-Dawley rats were divided into two experimental groups as follows: control group (n=30) and a Diabetes mellitus group (n=40) induced by injection of streptozotozin (STZ). The rats were investigated at 1, 4, 8, 12 and 16 weeks after STZ-treatment. By means of immunohistochemistry, the expression of Ets-1 in glomeruli was significantly increased in STZ-treated rat kidneys from week 1 (P<0.05) and reached the peak at week 4 (P<0.05), followed by a downward trend at subsequent time points. Similarly, the expression of Ets-1 in the tubulointerstitium was also markedly increased from week 1 (P<0.05) and reached a maximum at week 8 (P<0.05). By double immunostaining, Ets-1-positive cells were frequently found to co-express matrix metalloproteinase-2 (MMP-2) in STZ-treated rat kidneys. Increased expression of tissue inhibitor of metalloproteinase-2 (TIMP-2) coincided with increased expression of alpha-smooth muscle actin (alpha-SMA) in STZ-induced DN. A positive relationship was observed between the expression of Ets-1 in glomeruli or tubulointerstitium and the expression of MMP-2 (P<0.01; P<0.01, respectively) in STZ-treated rat kidneys. The ratio of MMP-2 and TIMP-2 in glomeruli or tubulointerstitium was negatively correlated with deposition of type IV collagen (P<0.01; P<0.01, respectively). These findings suggest that Ets-1 may play a critical role in fine-tuning matrix remodeling of STZ-induced DN.

The aim of the study was to explore the role of basic fibroblast growth factor (bFGF) in ovarian cancer progression. This was done by investigating the effects of bFGF on both the secretion of urokinase-type plasminogen activator (uPA) and the invasion of tumor cells in SKOV(3) ovarian cancer cells. Human ovarian cancer cell line SKOV(3) was cultured in vitro. The expression of uPA gene and protein was induced in SKOV(3) cells; the impact of bFGF on the expression of uPA gene in SKOV(3) cells was studied by RT-PCR, and the impact of bFGF on the expression of uPA protein was tested by ELISA. Ets-1 antisense oligonucleotides were transfected into SKOV(3) cells by liposome protocol. The effects of bFGF on Ets-1 expression and the invasion ability of SKOV(3) cells were determined both before and after exposure to different concentrations of bFGF for 24 h. The expression of both uPA gene and protein was induced in SKOV(3) cells, p < 0.05. The expression of uPA was suppressed by Ets-1 antisense oligonucleotides in SKOV(3) cells, p < 0.05. The invasion ability of SKOV(3) cells was increased by 2.3-fold, and this effect was also suppressed by Ets-1 antisense oligonucleotides. bFGF can enhance the invasion ability of ovarian cancer cells in vitro by inducing the expression of uPA, and this effect is also regulated by the transcription factor Ets-1.

Several ETS transcription factors, including MEF/ELF4 and ERG, can function as oncogenes and are overexpressed in human cancer. MEF cooperates in tumorigenesis in retroviral insertional mutagenesis-based mouse models of cancer and MEF is overexpressed in human lymphoma and ovarian cancer tissues via unknown mechanisms. ERG (Ets related gene) overexpression or increased activity has been found in various human cancers, including sarcomas, acute myeloid leukemia and prostate cancer, where the ERG gene is rearranged due to chromosomal translocations. We have been examining how MEF functions as an oncogene and recently showed that MEF can cooperate with H-Ras(G12V) and can inhibit both p53 and p16 expression thereby promoting transformation. In fact, in cells lacking p53, the absence of Mef abrogates H-Ras(G12V)-induced transformation of mouse embryonic fibroblasts, at least in part due to increased p16 expression. We discuss the known mechanisms by which the ETS transcription factors MEF and ERG contribute to the malignant transformation of cells.

The effect of interleukin-1 beta (IL-1) on expression of c-fos mRNA in isolated rat pancreatic islets was examined. Accumulation of c-fos mRNA was demonstrable after 30 min of exposure to IL-1, peaked by 60 min, and declined thereafter. Fluorescence-activated cell sorting (FACS) of dispersed islet cells was employed to localize the accumulation of c-fos mRNA to the beta-cell. Cycloheximide did not influence the induction of c fos mRNA by IL-1. Accumulation of c-fos mRNA therefore appears to be an early signal transduction event in the beta-cell and a component of the cellular mechanism(s) by which IL-1 influences beta-cell function.

The gene fusions between transmembrane protease serine 2 (TMPRSS2) and E26 (ETS) transcription factors are present in over 50% of patients with prostate cancer. TMPRSS2-ERG is the most common gene fusion type. The ERG overexpression induced by TMPRSS2-ERG gene fusion contributes to the development of prostate cancer. Both androgen receptor binding and genotoxic stress induce chromosomal proximity and TMPRSS2-ETS gene fusions. TMPRSS2-ERG gene fusion functions as a biomarker for prostate cancer, which can be easily detected in urine. This review focuses on the characteristics, oncogenic and rearranged mechanism, and clinical application of TMPRSS2-ETS gene fusions.

The microRNA miR-125b is dysregulated in various human cancers but its underlying mechanisms of action are poorly understood. Here, we report that miR-125b is downregulated in invasive breast cancers where it predicts poor patient survival. Hypermethylation of the miR-125b promoter partially accounted for reduction of miR-125b expression in human breast cancer. Ectopic restoration of miR-125b expression in breast cancer cells suppressed proliferation, induced G(1) cell-cycle arrest in vitro, and inhibited tumorigenesis in vivo. We identified the ETS1 gene as a novel direct target of miR-125b. siRNA-mediated ETS1 knockdown phenocopied the effect of miR-125b in breast cell lines and ETS1 overexpression in invasive breast cancer tissues also correlated with poor patient prognosis. Taken together, our findings point to an important role for miR-125b in the molecular etiology of invasive breast cancer, and they suggest miR-125b as a potential theranostic tool in this disease.

A suitable in vitro model system to investigate mechanisms of human prostate carcinogenesis is much needed. We have previously demonstrated that an immortal, but non-tumorigenic, human prostate epithelial cell line (267B(1)) can be malignantly transformed by the v-Ki-ras oncogene, and it can serve as a useful model for investigation of the progression steps of prostate carcinogenesis. In this study, we report for the first time the invasive/metastatic phenotype of the v-Ki-ras transformed 267B, cells (267B(1)/Ki-ras). In addition, comparing non-tumorigenic 267B, and metastatic tumorigenic 267B(1)/Ki-ras human prostate epithelial cell lines, we have found that expression of ETS-1 and ERGB mRNA was elevated to 2-5 fold in the metastatic and tumorigenic 267B(1)/Ki-ras cell line. A specific ETS-1 monoclonal antibody E44 also revealed that the expression of ETS-1 protein level in 267B(1)/Ki-ras cell line was higher than those in 267B, cell line. However, other members of the ETS gene family such as ETS-2, GABP alpha and their mRNA expression levels were similar in both cell lines. The activation of MAP kinase, a downstream target for Ki-ras, was also shown. The expression of urokinase plasminogen activator (u-PA) was also increased in the metastatic 267B(1)/Ki-ras line. An obvious capability of invasion was observed in the 267B(1)/Ki-ras cell line, but not in the 267B(1) line using BioCoat Matrigel invasion chamber assay system. The present study has provided evidence that the v-Ki-ras oncogene may activate the nuclear target gene, ETS-1 gene, to mediate tumorigenic and metastatic capacity of the v-Ki-ras transformed prostate epithelial cells.

ETS-1 protooncogene is an important transcription factor that plays a role in the regulation of physiological processes, such as cell proliferation and differentiation. ETS-1 is thought to be related to the growth of carcinoma cells by its regulation of the transcription of matrix metalloproteinases and urokinase-type plasminogen activator. In this study, we aimed to investigate the expression pattern of ETS-1 oncoprotein in urothelial carcinomas of the urinary bladder and determine its relationship with histopathologic parameters, including tumor grade and stage. One hundred six specimens of urothelial carcinoma and a total of 14 normal urothelium were analyzed immunohistochemically with anti-ETS-1 monoclonal antibody. The normal urothelium showed positive ETS-1 immunostaining. ETS-1 expression remained high in low-grade and noninvasive tumors, whereas it frequently decreased in high-grade or invasive carcinomas. Interestingly, ETS-1 was highly expressed in the basal cell layer of the noninvasive urothelial carcinomas. ETS-1 expression showed a strong negative correlation with the tumor grade (P<0.001; r, -0.67) and stage (P<0.001; r, -0.75). The nonmuscle-invasive tumors (pTa+pT1) and noninvasive tumors (pTa) had significantly higher ETS-1 expression than the muscle-invasive tumors (pT2; P<0.001) and invasive tumors (pT1+pT2; P<0.001), respectively. Results of our study show that decreased ETS-1 expression is significantly associated with high grade and advanced stage in urothelial carcinomas of the urinary bladder, and that the downregulation of ETS-1 expression may be a marker of the aggressiveness of such malignancies.

The ets proto-oncogene family is a group of sequence-related genes whose normal cellular function is unknown. In a study of cellular proteins involved in the transcriptional regulation of murine retroviruses in T lymphocytes, we have discovered that a member of the ets gene family encodes a sequence-specific DNA-binding protein. A mouse ets-1 cDNA clone was obtained by screening a mouse thymus cDNA expression library with a double-stranded oligonucleotide probe representing 20 bp of the Moloney murine sarcoma virus (MSV) long terminal repeat (LTR). The cDNA sequence has an 813-bp open reading frame (ORF) whose predicted amino acid sequence is 97.6% identical to the 272 carboxy-terminal amino acids of the human ets-1 protein. The ORF was expressed in bacteria, and the 30-kD protein product was shown to bind DNA in a sequence-specific manner by mobility-shift assays, Southwestern blot analysis, and methylation interference. A mutant LTR containing four base pair substitutions in the ets-1 binding site was constructed and was shown to have reduced binding in vitro. Transcriptional efficiency of the MSV LTR promoter containing this disrupted ets-1 binding site was compared to the activity of a wild-type promoter in mouse T lymphocytes in culture, and 15- to 20-fold reduction in expression of a reporter gene was observed. We propose that ets-1 functions as a transcriptional activator of mammalian type-C retroviruses and speculate that ets-related genes constitute a new group of eukaryotic DNA-binding proteins.

MicroRNAs-221 and -222 are highly upregulated in several solid tumors, including melanomas. We demonstrate that the proto-oncogene ETS-1, involved in the pathogenesis of cancers of different origin, is a transcriptional regulator of miR-222 by direct binding to its promoter region. Differently from 293FT cells or early stage melanomas, where unphosphorylated ETS-1 represses miR-222 transcription, in metastatic melanoma the constitutively Thr-38 phosphorylated fraction of ETS-1 induces miR-222. Despite its stepwise decreased expression along with melanoma progression, the oncogenic activity of ETS-1 relies on its RAS/RAF/ERK-dependent phosphorylation status more than on its total amount. To close the loop, we demonstrate ETS-1 as a direct target of miR-222, but not miR-221, showing the novel option of their uncoupled functions. In addition, a spatial redistribution of ETS-1 protein from the nucleus to the cytoplasm is also evidenced in advanced melanoma cells. Finally, in vivo studies confirmed the contribution of miR-222 to the increased invasive potential obtained by ETS- silencing.

Five years of tamoxifen reduces breast cancer risk by nearly 50% but is associated with significant side effects and toxicities. A better understanding of the direct and indirect effects of tamoxifen in benign breast tissue could elucidate new mechanisms of breast carcinogenesis, suggest novel chemoprevention targets, and provide relevant early response biomarkers for phase II prevention trials. Seventy-three women at increased risk for breast cancer were randomized to tamoxifen (20 mg daily) or placebo for 3 months. Blood and breast tissue samples were collected at baseline and posttreatment. Sixty-nine women completed ALL study activities (37 tamoxifen and 32 placebo). The selected biomarkers focused on estradiol and IGFs in the blood; DNA methylation and cytology in random periareolar fine-needle aspirates; and tissue morphometry, proliferation, apoptosis, and gene expression (microarray and reverse transcriptase PCR) in the tissue core samples. Tamoxifen downregulated Ets oncogene transcription factor family members ETV4 and ETV5 and reduced breast epithelial cell proliferation independent of CYP2D6 genotypes or effects on estradiol, ESR1, or IGFs. Reduction in proliferation was correlated with downregulation of ETV4 and DNAJC12. Tamoxifen reduced the expression of ETV4- and ETV5-regulated genes implicated in epithelial-stromal interaction and tissue remodeling. Three months of tamoxifen did not affect breast tissue composition, cytologic atypia, preneoplasia, or apoptosis. A plausible mechanism for the chemopreventive effects of tamoxifen is restriction of lobular expansion into stroma through downregulation of ETV4 and ETV5. The human equivalent of murine multipotential progenitor cap cells of terminal end buds may be the primary target.

ETS-1 protein is one of the key regulators in tumor invasion and progression. We aimed to evaluate the role of ETS-1 in the invasiveness and progression of laryngeal squamous carcinoma, as well as to determine the correlations between clinicopathological characteristics and expression of this molecule. We assessed the levels of ETS-1 in a total of 96 laryngeal specimens of varying degrees of dysplasia, microinvasive squamous carcinoma (8), and invasive squamous carcinoma (60), using normal mucosal epithelium (10) as a positive control. The relationship between ETS-1 expression and clinicopathological parameters of laryngeal carcinoma was also analyzed. We found a significantly higher ETS-1 expression in invasive laryngeal squamous cell carcinomas than in dysplasia (P<0.001). A correlation between ETS-1 expression scores and grade was detected - T factor, stage, cartilage invasion, lymph node metastasis, as well as depth of invasion in laryngeal tumors. Our study is the first to demonstrate that ETS-1 expression is significantly increased in invasive carcinoma, but it is absent in low-moderate grade laryngeal dysplasia and non-neoplastic laryngeal mucosa. This data suggest that ETS-1 expression may play an important role in tumor invasion, and may function in the initiation of the invasive process in laryngeal squamous cell carcinoma.

expression of viral proteins causes important epigenetic changes leading to abnormal cell growth. Whether viral proteins directly target histone methyltransferases (HMTs), a key family enzyme for epigenetic regulation, and modulate their enzymatic activities remains elusive. Here we show that the E6 proteins of both low-risk and high-risk human papillomavirus (HPV) interact with three coactivator HMTs, CARM1, PRMT1 and SET7, and downregulate their enzymatic activities in vitro and in HPV-transformed HeLa cells. Furthermore, these three HMTs are required for E6 to attenuate p53 transactivation function. Mechanistically, E6 hampers CARM1- and PRMT1-catalyzed histone methylation at p53-responsive promoters, and suppresses the binding of p53 to chromatinized DNA independently of E6-mediated p53 degradation. p53 pre-methylated at lysine-372 (p53K372 mono-methylation) by SET7 protects p53 from E6-induced degradation. Consistently, E6 downregulates p53K372 mono-methylation and thus reduces p53 protein stability. As a result of the E6-mediated inhibition of HMT activity, expression of p53 downstream genes is suppressed. Together, our results not only reveal a clever approach for the virus to interfere with p53 function, but also demonstrate the modulation of HMT activity as a novel mechanism of epigenetic regulation by a viral oncoprotein.

The aberrant expression of an oncogenic ETS transcription factor is implicated in the progression of the majority of prostate cancers, 40% of melanomas, and most cases of gastrointestinal stromal tumor and Ewing s sarcoma. Chromosomal rearrangements in prostate cancer result in overexpression of any one of four ETS transcription factors. How these four oncogenic ETS genes differ from the numerous other ETS genes expressed in normal prostate and contribute to tumor progression is not understood. We report that these oncogenic ETS proteins, but not other ETS factors, enhance prostate cell migration. Genome-wide binding analysis matched this specific biological function to occupancy of a unique set of genomic sites highlighted by the presence of ETS- and AP-1-binding sequences. ETS/AP-1-binding sequences are prototypical RAS-responsive elements, but oncogenic ETS proteins activated a RAS/MAPK transcriptional program in the absence of MAPK activation. Thus, overexpression of oncogenic ETS proteins can replace RAS/MAPK pathway activation in prostate cells. The genomic description of this ETS/AP-1-regulated, RAS-responsive, gene expression program provides a resource for understanding the role of these ETS factors in both an oncogenic setting and the developmental processes where these genes normally function.

The chicken cellular c-ets-1 locus encodes for two related proteins generated by alternative splicing: the widely expressed p54c-ets-1 protein and the cellular homolog of the v-ets-encoded domain of the E26-transforming protein P135gag-myb-ets, p68c-ets-1 which has been found so far only in the spleen. We have prepared a new site specific antiserum directed against the amino-terminus of p68c-ets-1, which is highly hydrophobic by contrast to the hydrophilic NH2 terminus of p54c-ets-1. This antiserum specifically immunoprecipitated p68c-ets-1 and P135gag-myb-ets only in denaturing and reducing conditions. Despite these biochemical differences at their amino-terminal parts, p68c-ets-1, as p54c-ets-1, is a nuclear protein able to bind to DNA in vitro. Unlike p54c-ets-1 which is expressed at high levels in T- and B-lymphoid cells, p68c-ets-1 is not expressed in lymphoid cells of the spleen. Thus, the two c-ets-1 encoded proteins, although both exhibiting DNA-binding properties in vitro, display differences both in the nature of their specific NH2 termini, and in their level and pattern of expression.

BGC-Ha-ras, isolated from the human gastric cancer cell line BGC-823 and activated by point mutation, is a transforming oncogene. To study whether this oncogene can still be located in the original position of chromosome 11 of BGC-823 cell line, chromosomal in situ hybridization method was used. The results showed that 31% silver grains were localized on chromosome 11, 83% of which on the 11p15 region, and in addition, 10% grains on distal end of the short arm of the other abnormal chromosome. Such results are significant in further study of the relation between gene localization and carcinogenesis.

The c-ets-1 proto-oncogene and the related c-ets-2 gene encode related nuclear chromatin-associated proteins which bind DNA in vitro. To investigate the possibility that Ets1 and Ets2 are transcriptional activators, we analyzed the ability of these proteins to trans-activate promoter/enhancer sequences in transient co-transfection experiments. A CAT construct driven by the long terminal repeat of the human T cell leukemia virus, HTLV-1 was found to be trans-activated by both Ets1 and Ets2 in NIH3T3 and HeLa cells. The increased levels of CAT activity were paralleled by increased levels of correctly initiated CAT mRNA. Mutant Ets1 proteins unable to accumulate in the nucleus were found to be inactive. An ets-responsive sequence between positions -117 and -160 of the LTR was identified by analyses of a series of 5 deletion mutants of the HTLV-1 LTR and of dimerized versions of specific motifs of the LTR enhancer region. Using a gel shift binding assay, Ets1 was found to bind specifically to an oligonucleotide corresponding to region -117 to -160. This sequence, which also contributes to Tax1 responsiveness of the HTLV-1 LTR, is characterized by the presence of four repeats of a pentanucleotide sequence of the type CC(T/A)CC. Competition experiments show that integrity of repeats 1 and 4 is important for Ets1 binding. These results show that Ets1 and Ets2 are sequence-specific transcriptional activators. In view of the high level expression of Ets1 in lymphoid cells, Ets1 could be part of the transcription complex which mediates the response to Tax1 and the control of HTLV-1 replication. More generally, Ets1 and Ets2 could regulate transcription of cellular genes.

The homologous ETV1, ETV4 and ETV5 proteins form the PEA3 subfamily of ETS transcription factors. In Ewing tumors, chromosomal translocations affecting ETV1 or ETV4 are an underlying cause of carcinogenesis. Likewise, chromosomal rearrangements of the ETV1, ETV4 or ETV5 gene occur in prostate tumors and are thought to be one of the major driving forces in the genesis of prostate cancer. In addition, these three ETS proteins are implicated in melanomas, breast and other types of cancer. Complex posttranslational modifications govern the activity of PEA3 factors, which can promote cell proliferation, motility and invasion. Here, we review evidence for a role of ETV1, 4 and 5 as oncoproteins and describe modes of their action. Modulation of their activation or interaction with cofactors as well as inhibiting crucial target gene products may ultimately be exploited to treat various cancers that are dependent on the PEA3 group of ETS transcription factors.

MicroRNAs (miRs) are important regulators of gene expression in normal physiology and disease, and are widely misexpressed in cancer. A number of studies have identified miR-21 as an important promoter of oncogenesis. However, as is true of most miRs, the mechanisms behind the aberrant expression of miR-21 in cancer are poorly understood. Herein, we examine the regulation of miR-21 expression in colorectal cancer (CRC) cells by the oncogenic epidermal growth factor (EGF)/Ras pathway and by Ets transcription factors, modulators of epithelial oncogenesis that are frequently misexpressed in CRC. We show that EGF/Ras efficiently induces the miR-21 primary transcript, but this does not rapidly and simply translate into higher mature miR-21 levels. Rather, induction of mature miR-21 by constitutive activation of this pathway is slow, is associated with only minimal activation of mitogen-activated protein kinase, and may involve stimulation of post-transcriptional processing by mechanisms other than Dicer stabilization. We further identify Ets transcription factors as modifiers of miR-21 expression in CRC. The effects of Ets factors on miR-21 expression are cell context-dependent, and appear to involve both direct and indirect mechanisms. The Ets factor Pea3 emerges from our studies as a consistent repressor of miR-21 transcription. Overall, our studies identify a complex relationship between oncogenic pathways and steady-state miR-21 levels in CRC, and highlight the need for greater understanding of the control of miR expression in cancer and other disease states.

The specific chromosomal rearrangement t(11;19)(q23;p13) has been identified as a nonrandom chromosomal rearrangement in acute leukemia. The breakpoint, 11q23, coincides with the ets-1 oncogene locus. However, only very few studies have been done to verify the genomic alteration and transposition of ets-1 in the t(11;19) chromosomal rearrangement. In the present study, we identified the t(11;19)(q23;p13) translocation in two acute leukemic cases. One of the cases, biphenotypic leukemia, has been followed thoroughly. An abnormal karyotype was identified in the patient s blood and marrow samples at diagnosis and at relapse, while only normal karyotypes were identified at remission. In situ hybridization of chromosomal preparations with the ets-1 probe pHE5.4 resulted in silver grains nonrandomly localized to 19p13 in the metaphase spreads prepared from the blood sample taken at relapse, while no detectable grains were found on chromosome 19p13 in a sample taken at remission. To determine if genomic alterations of ets-1 are associated with this translocation, Southern blot hybridizations with the pHE5.4 probe were performed on deoxyribonucleic acid (DNA) isolated from blood or marrow samples of the patient at remission and relapse as well as on DNA from a disease-free normal control. Any DNA digested with AvaII, SstI, XbaI, and Bam HI, followed by hybridization with pHE5.4, demonstrated no genomic alterations or amplification of the ets-1 oncogene. Our study indicates that the ets-1 oncogene is transposed in the t(11;19) translocation without detectable alteration at the DNA level. The absence of ets-1 amplification in t(11;19) and its presence in the t(4,11) and t(9;11) translocations demonstrated by others suggests the possible existence of different molecular mechanisms involving the ets-1 oncogene in the pathogenesis of these leukemias.

MicroRNAs may function to promote or suppress tumor development, depending on the cellular context. The important role of microRNAs in regulating molecular pathways underlying tumorigenesis has been emphasized in hepatocellular carcinoma (HCC). MicroRNAs regulate gene expression via post-transcriptional mechanisms by inhibiting translation or by degrading mRNA. In this study, we show that microRNA-1 (miR-1) and microRNA-499 (miR-499) are capable of repressing the expression of the ets1 proto-oncogene, which plays a fundamental role in the extracellular matrix (ECM) degradation, a process required for tumor cell invasion and migration. We used luciferase reporter assays to demonstrate that miR-1 and miR-499 target the 3 untranslated region (UTR) of ets1. Overexpression of miR-1 and miR-499 in HepG2 cells led to downregulation of ets1 mRNA and protein as assessed by quantitative reverse transcription PCR and western blot analysis. Furthermore, overexpression of miR-1 and miR-499 inhibited the invasion and migration of HepG2 cells in matrigel invasion and transwell migration assays, respectively. These results suggest that miR-1 and miR-499 may play an important role in the pathogenesis of HCC by regulating ets1.

The proto-oncogene ets-1 is a member of the ets family of genes that share homology with the viral oncogene, v-ets, of the avian leukemia virus E26. By using expression vectors, we demonstrate that the ets-1 gene transforms NIH3T3 cells and the ets-1 transfected cells form colonies in soft agar and induce tumors in nude mice. We have also determined that the ets-1 protein contains homology with the helix-loop-helix motif of the HLH family proteins, but lacks the basic domain upstream of helix I. Transfection of the NIH3T3 cells with ets-1 vectors results in the activation of the endogenous ets-1 gene. Using hybridization probes that can distinguish between transcripts from endogenous and exogenous templates, we show that the endogenous ets-1 gene is activated by the expression of the transfected exogenous ets-1. In contrast, the expression of transfected ets-2 has no effect on the endogenous ets-1 gene expression. The results indicate that the ets-1 proto-oncogene is positively autoregulated by its product.

INTRODUCTION: The Ets-1 transcription factor is a candidate breast cancer oncogene that regulates the expression of genes involved in tumor progression and metastasis. Ets-1 signaling has also been linked to the development of a basal-like breast cancer phenotype. We recently described a nitric oxide (NO)-induced gene signature that is associated with poor disease outcome in estrogen receptor-negative (ER-) breast cancer and contains both stem cell-like and basal-like components. Thus, we examined the role of Ets-1 in NO signaling and NO-induced phenotypes in ER- human breast cancer cells. METHODS: Promoter region analyses were performed on genes upregulated in inducible nitric oxide synthase (NOS2) high expressing tumors for Ets-binding sites. In vitro mechanisms were examined in human basal-like breast cancer cells lines. NO signaling effects were studied using either forced NOS2 expression or the use of a chemical NO-donor, diethlylenetriamine NONOate (DETANO). RESULTS: Promoter region analysis of genes that are up-regulated in human ER-negative breast tumors with high NOS2 expression revealed that the Ets-binding sequence is the only common promoter element present in ALL of these genes, indicating that Ets-1 is the key transcriptional factor down-stream of oncogenic NOS2-signaling. Accordingly, both forced NOS2 over-expression and exposure to NO-donors resulted in significant Ets-1 transcriptional activation in ER- breast cancer cells. Functional studies showed that NO activated Ets-1 transcriptional activity via a Ras/MEK/ERK signaling pathway by a mechanism that involved Ras S-nitrosylation. RNA knock-down of Ets-1 suppressed NO-induced expression of selected basal-like breast cancer markers such as P-cadherin, S100A8, IL-8 and alphabeta-crystallin. Additionally, Ets-1 knock-down reduced NO-mediated cellular proliferation, matrix metalloproteinase and cathepsin B activities, as well as matrigel invasion. CONCLUSIONS: These data show that Ets-1 is a key transcriptional mediator of oncogenic NO signaling that promotes the development of an aggressive disease phenotype in ER- breast cancer in an Ets-1 and Ras-dependent manner, providing novel clues of how NOS2 expression in human breast tumors is functionally linked to poor patient survival.

miR-199a-5p plays a critical role in controlling cardiomyocyte survival. However, its significance in endothelial cell biology remains ambiguous. Here, we report the first evidence that miR-199a-5p negatively regulates angiogenic responses by directly targeting v-ets erythroblastosis virus E26 oncogene homolog 1 (Ets-1). Induction of miR-199a-5p in human dermal microvascular endothelial cells (HMECs) blocked angiogenic response in Matrigel(R) culture, whereas miR-199a-5p-deprived cells exhibited enhanced angiogenesis in vitro. Bioinformatics prediction and miR target reporter assay recognized Ets-1 as a novel direct target of miR-199a-5p. Delivery of miR-199a-5p blocked Ets-1 expression in HMECs, whereas knockdown endogenous miR-199a-5p induced Ets-1 expression. Matrix metalloproteinase 1 (MMP-1), one of the Ets-1 downstream mediators, was negatively regulated by miR-199a-5p. Overexpression of Ets-1 not only rescued miR-199a-5p-dependent anti-angiogenic effects but also reversed miR-199a-5p-induced loss of MMP-1 expression. Similarly, Ets-1 knockdown blunted angiogenic response and induction of MMP-1 in miR-199a-5p-deprived HMECs. Examination of cutaneous wound dermal tissue revealed a significant down-regulation of miR-199a-5p expression, which was associated with induction of Ets-1 and MMP-1. Mice carrying homozygous deletions in the Ets-1 gene exhibited blunted wound blood flow and reduced abundance of endothelial cells. Impaired wound angiogenesis was associated with compromised wound closure, insufficient granulation tissue formation, and blunted induction of MMP-1. Thus, down-regulation of miR-199a-5p is involved in the induction of wound angiogenesis through derepressing of the Ets-1-MMP1 pathway.

Recent evidence shows that v-ets erythroblastosis virus E26 oncogene homolog 1 (Ets1) is implicated in tumor development and progression. However, the clinical potentials and underlying mechanisms of Ets1 in gastric cancer progression and metastasis remain largely unknown. In this study, Ets1 immunostaining was identified in 56 of 84 (66.7%) gastric cancer tissues, which was correlated with tumor invasion and metastasis. In gastric cancer specimens and cell lines, miRNA-145 (miR-145) was downregulated and inversely correlated with Ets1 expression. Bioinformatics analysis and luciferase reporter assay revealed that miR-145 directly targeted the 3 -untranslated region (3 -UTR) of Ets1 mRNA. Overexpression or knockdown of miR-145 responsively altered both the mRNA and protein levels of Ets1 and its downstream genes, matrix metalloproteinase (MMP-1)-1 and -9, in gastric cancer cell lines SGC-7901 and MKN-45. Ectopic expression of miR-145 suppressed the invasion, metastasis, and angiogenesis of SGC-7901 and MKN-45 cells in vitro and in vivo. In addition, the effects of miR-145 on Ets1 expression, migration, invasion, and angiogenesis were rescued by restoration of Ets1 expression in these cells. Furthermore, anti-miR-145 inhibitor promoted the migration, invasion, and angiogenesis, whereas siRNA-mediated Ets1 knockdown phenocopied the effects of miR-145 overexpression in gastric cancer cells. These results show that miR-145 suppresses Ets1 expression via the binding site in the 3 -UTR, thus inhibiting the invasion, metastasis, and angiogenesis of gastric cancer cells.

We previously reported overexpression of Prostate derived Ets transcription factor (PDEF) in breast cancer and its role in breast cancer progression, supporting PDEF as an attractive target in this cancer. The goal of this research was to identify specific PDEF induced molecules that, like PDEF, show overexpression in breast tumors and a role in breast tumor progression. PDEF expression was down regulated by shRNA in MCF-7 human breast tumor cell line, and probes from PDEF down-regulated and control MCF-7 cells were used to screen the HG-U133A human gene chips. These analyses identified 1318 genes that were induced two-fold or higher by PDEF in MCF-7 cells. Further analysis of three of these genes, namely CEACAM6, S100A7 and B7-H4, in relation to PDEF in primary breast tumors showed that in 82% of ER+, 67% of Her2 overexpressing and 24% of triple-negative breast tumors both PDEF and CEACAM6 expression was elevated 10-fold or higher in comparison to normal breast tissue. Overall, 72% (94 of 131) of the primary breast tumors showed 10-fold or higher expression of both PDEF and CEACAM6. In contrast, S100A7 and B7-H4 failed to show concordant elevated expression with PDEF in primary tumors. To determine the significance of elevated PDEF and CEACAM6 expression to tumor phenotype, their expression was down regulated by specific siRNAs in human breast tumor cell lines. This resulted in the loss of viability of tumor cells in vitro, supporting an oncogenic role for both PDEF and CEACAM6 in breast cancer. Together, these findings show that PDEF-CEACAM6 is a highly active oncogenic axis in breast cancer and suggest that targeting of these molecules should provide novel treatments for most breast cancer patients.

We prepared antisera to three distinct portions of the v-ets oncogene of the avian leukemia virus E26. An antiserum directed against the middle v-ets-encoded domain identifies in different chicken cell lines and normal tissues a c-ets-encoded protein of Mr 54,000 (P54c-ets) and three proteins of Mr 60,000 62,000 and 64,000 partially related to P54c-ets. Antisera directed against the aminoterminal v-ets-encoded domain failed to precipitate P54c-ets or P60/P64. Thus, the E26 specific v-ets oncogene displays a complex structure that includes several distinct portions, the genetic origin of which could be different.

We have cloned part of the ETS 1 proto-oncogene and demonstrated the presence of two polymorphic Sst I restriction sites. A probe derived from one of our clones revealed the presence of 8.3 kb, 9.5 kb and/or 11.5 kb fragments on Southern blots of human DNA samples. The relative frequencies of these alleles appear to be significantly different between Saudi and Western populations, but there are no apparent differences in these frequencies between Saudi non-leukemic and leukemic individuals.

The ETS-family transcription factors Ets1 and Ets2 are evolutionarily conserved effectors of the RAS/ERK signaling pathway, but their function in Ras cellular transformation and biology remains unclear. Taking advantage of Ets1 and Ets2 mouse models to generate Ets1/Ets2 double knockout mouse embryonic fibroblasts, we demonstrate that deletion of both Ets1 and Ets2 was necessary to inhibit HrasG12V induced transformation both in vitro and in vivo. HrasG12V expression in mouse embryonic fibroblasts increased ETS1 and ETS2 expression and binding to cis-regulatory elements on the c-Myc proximal promoter, and consequently induced a robust increase in MYC expression. The expression of the oncogenic microRNA 17-92 cluster was increased in HrasG12V transformed cells, but was significantly reduced when ETS1 and ETS2 were absent. MYC and ETS1 or ETS2 collaborated to increase expression of the oncogenic microRNA 17-92 cluster in HrasG12V transformed cells. Enforced expression of exogenous MYC or microRNA 17-92 rescued HrasG12V transformation in Ets1/Ets2-null cells, revealing a direct function for MYC and microRNA 17-92 in ETS1/ETS2-dependent HrasG12V transformation.

The molecular response to hypoxia is a critical cellular process implicated in cancer, and a target for drug development. The activity of the major player, HIF1alpha, is regulated at different levels by various factors, including the transcription factor ELK3. The molecular mechanisms of this intimate connection remain largely unknown. Whilst investigating global ELK3-chromatin interactions, we uncovered an unexpected connection that involves the microRNA hsa-miR-155-5p, a hypoxia-inducible oncomir that targets HIF1alpha. One of the ELK3 chromatin binding sites, detected by Chromatin Immuno-Precipitation Sequencing (ChIP-seq) of normal Human Umbilical Vein Endothelial Cells (HUVEC), is located at the transcription start site of the MIR155HG genes that expresses hsa-miR-155-5p. We confirmed that ELK3 binds to this promoter by ChIP and quantitative polymerase chain reaction (QPCR). We showed that ELK3 and hsa-miR-155-5p form a double-negative regulatory loop, in that ELK3 depletion induced hsa-miR-155-5p expression and hsa-miR-155-5p expression decreased ELK3 expression at the RNA level through a conserved target sequence in its 3 -UTR. We further showed that the activities of hsa-miR-155-5p and ELK3 are functionally linked. pathway analysis indicates that both factors are implicated in related processes, including cancer and angiogenesis. Hsa-miR-155-5p expression and ELK3 depletion have similar effects on expression of known ELK3 target genes, and on in-vitro angiogenesis and wound closure. Bioinformatic analysis of cancer RNA-seq data shows that hsa-miR-155-5p and ELK3 expression are significantly anti-correlated, as would be expected from hsa-miR-155-5p targeting ELK3 RNA. Finally, hypoxia (0% oxygen) down-regulates ELK3 mRNA in a microRNA and hsa-miR-155-5p dependent manner. These results tie ELK3 into the hypoxia response pathway through an oncogenic microRNA and into a circuit implicated in the dynamics of the hypoxic response. This crosstalk could be important for the development of new treatments for a range of pathologies.

T-cell lymphomas induced in rats by Moloney murine leukemia virus acquire increasing numbers of proviruses in their genome during tumor progression in vivo and passage of tumor cells in vitro. To determine whether the proviruses progressively acquired during tumor progression play a causal role in this process, we cloned one of them from a cell line derived from the primary tumor 2772. A probe from the cellular DNA flanking the provirus was used to analyze 79 DNA samples from primary tumor tissues of 28 tumor-bearing rats and 80 DNA samples from 30 independent tumor cell lines. This analysis revealed a rearrangement in this region in the primary tumor derived from the thymus of one animal but not in a clone of the same tumor segregating in the spleen. Of the cell line DNA samples, three carried a provirus in this region. Two of these integration events had occurred independently in two clonally related sublines derived from tumor 2772, and they were followed by rapid selection in culture. On the basis of these findings this locus was named Tpl-1 (tumor progression locus 1). The Tpl-1 locus was mapped to rat chromosome 8 and to mouse chromosome 9 at a genetic distance of 1.2 +/- 0.9 centimorgans from the Ets-1 protooncogene. Although the genetic distance between Tpl-1 and Ets-1 indicates that they are different genes, analysis of Tpl-1 cDNA clones revealed that the two are closely related.

Gastrin-releasing peptide receptor (GRPR) is known to be overexpressed in several human malignancies, including prostate cancer, and has been implicated in multiple important neoplastic signaling pathways. We recently have shown that GRPR is an ERG and ETV1 target gene in prostate cancer, using a genome-wide scale and exon-level expression microarray platform. Due to its cellular localization, the relevance of its function and the availability of blocking agents, GRPR seems to be a promising candidate as therapeutic target. Our present work shows that effective knockdown of GRPR in LNCaP and VCaP cells attenuates their malignant phenotype by decreasing proliferation, invasion and anchorage-independent growth, while increasing apoptosis. Using an antibody microarray we were able to validate known and identify new targets of GRPR pathway, namely AKT1, PKCepsilon, TYK2 and MST1. Finally, we show that overexpression of these GRPR targets is restricted to prostate carcinomas harboring ERG and/or ETV1 rearrangements, establishing their potential as therapeutic targets for these particular molecular subsets of the disease.

BACKGROUND: The aim of this study was to investigate if the expression of CD105 and Ets-1 was predictive of aggressive biologic behavior of non-melanoma skin cancers (NMSC) and to evaluate indicators of local recurrence. PATIENTS AND METHODS: A total of 144 patients with NMSC were included in the current study. Surgical specimens were independently examined for diagnosis confirmation and immunohistochemical expression of Ets-1 and CD105 by two dermatopathologists. RESULTS: The most common tumor type was basal cell carcinoma (n = 76), followed by squamous cell carcinomas (SCC) (n = 65). The most common anatomic location was the head and neck area (n = 115). The follow-up was > 2 years in ALL examined cases. A statistically significant correlation was found between tumor local recurrence and age (P = 0.03), Ets-1 expression (P < 0.0001) and CD105 expression (P < 0.0001). CONCLUSIONS: Our data confirm that both Ets-1 and CD105 show promise as prognostic markers for local recurrence of NMSC. However, this statement is made with caution, and additional studies, with larger populations, are necessary to examine the correlation between these two markers and local recurrence. A better understanding of the pathogenesis of local recurrence in primary NMSC may result in potential therapeutic interventions.

The neural crest (NC) is a transient, migratory cell population that differentiates into a large variety of tissues including craniofacial cartilage, melanocytes, and peripheral nervous system. NC is initially induced at the border of neural plate and non-neural ectoderm by balanced regulation of multiple signaling pathways among which an intermediate bone morphogenetic protein (BMP) signaling is essential for NC formation. ets1, a proto-oncogene playing important roles in tumor invasion, has also been implicated in delamination of NC cells. In this study, we investigated Ets1 function in NC formation using Xenopus. Overexpression of ets1 repressed NC formation through down-regulation of BMP signaling. Moreover, ets1 repressed the BMP-responsive gene id3 that is essential for NC formation. Conversely, overexpression of id3 can partially rescue the phenotype of NC inhibition induced by ectopic ets1. Mechanistically, we found that Ets1 binds to id3 promoter as well as histone deacetylase 1, suggesting that Ets1 recruits histone deacetylase 1 to the promoter of id3, thereby inducing histone deacetylation of the id3 promoter. Thus, our studies indicate that Ets1 regulates NC formation through attenuating BMP signaling epigenetically.

Field inversion gel electrophoresis of DNA from a leukemic clone characterized by the t(11;19)(q23;p13) allowed us to exclude any genomic rearrangement within more than 900 kb of DNA encompassing ETS1 on chromosome 11. Although ETS1 was moved to the derivative chromosome 19 as a result of the t(11;19), we conclude that this oncogene is not close to the chromosome 11 breakpoint and is unlikely to be involved in this leukaemia.

c-ets-1 and c-ets-2 are 2 proto-oncogenes known to be possibly involved in some human myelomonocytic leukemias. However, very few studies concern c-ets-1 and c-ets-2 RNA expression in human hematologic malignancies. We have studied 18 leukemic patients, and 10 cell lines for their ets RNA contents. c-ets-1 was strongly expressed in 5 and c-ets-2 in 8 of the 18 patients. ALL the cell lines expressed both c-ets-1 and c-ets-2 RNAs. This expression was highly variable from one patient to another, and from one cell line to another, regardless of the cellular leukemia subtype. The variability of this expression in patients may reflect differences in the proliferative potential of leukemic cells.

Two overlapping c-ets-1 cDNA clones were isolated which contained the alpha and beta genomic sequences homologous to the 5 end of v-ets not detected in the previously described c-ets RNA species or proteins. Nucleotide sequencing demonstrated that these cDNAs corresponded to the splicing of alpha and beta to a common set of 3 exons (a through F) already found in the p54c-ets-1 mRNA. They contained an open reading frame of 1,455 nucleotides which could encode a polypeptide of 485 amino acids with a predicted molecular mass of 53 kilodaltons. However, when expressed in COS-1 cells, the cDNAs directed the synthesis of a protein with an apparent molecular mass in sodium dodecyl sulfate-polyacrylamide gel electrophoresis of 68 kilodaltons, p68c-ets-1, comigrating with a protein expressed at low levels in normal chicken spleen cells. These two proteins were shown to be identical by partial digestion with protease V8. Northern (RNA) blot hybridization analysis with the p68c-ets-1 -specific sequence and RNase protection experiments showed that the corresponding mRNA was expressed in normal chicken spleen and not in normal chicken thymus or in various T lymphoid cell lines. Thus, two closely related proteins, having distinct amino-terminal parts, are generated within the same locus by alternative addition of different 5 exons, alpha and beta or I54, respectively, onto a common set of 3 exons (a to F). Finally, we demonstrate that an aberrant splicing event between a cryptic splice donor site in c-myb exon E6 and the normal splice acceptor site of c-ets-1 exon alpha involved in the genesis of the E26 myb-ets sequence.

We have recently cloned a novel human gene named erg related to the ets oncogene. In this report we describe the expression of the erg gene product in E. coli under the control of the tightly-regulated phage lambda PL promoter. Two expression vectors were utilized for the synthesis of the cII-erg and erg-fusion proteins. ALL these products were recognized by peptide antibodies prepared against the predicted amino acid sequences of erg and the Hu-ets-2 domain which shares homology with erg.

Twenty four samples of DNA from 23 unrelated individuals were analyzed for the presence of a novel restriction fragment length polymorphism (RFLP) involving the proto-oncogene ETS-1 at an Xba I site. Four samples from unrelated individuals lacked an Xba I site, giving rise to a longer restriction fragment detectable by Southern analysis; two samples were from normal tissue, and two were from acute myelogenous leukemic blasts. Thus, no association could be found between the RFLP and disease among the individuals studied. Pedigree analysis of another cohort demonstrated Mendelian inheritance consistent with a somatic polymorphism. The practical applications of RFLP analysis in clinical and research settings, and the usefulness of this Xba I RFLP in the study of hematologic malignancies because of its location in 11q23, are discussed.

The avian retrovirus E26 is unique among acute leukemia viruses in its ability to induce transformation of cells belonging to either the myeloid or erythroid lineage. The genome of E26 carries two oncogenes, v-myb and v-ets, that are derived from distinct cellular loci, c-myb and c-ets. We have constructed a plasmid vector that allows expression of part of the coding region of v-ets in a bacterial host. Antisera to the bacterially synthesized ets protein specifically precipitated the E26-encoded P135gag-myb-ets transforming protein. These antisera permitted us to identify a chicken c-ets-encoded protein of Mr 54,000 (P54c-ets) that shares 7 out of 10 of its major [35S]methionine-containing tryptic peptides with the v-ets-encoded domain of P135gag-myb-ets. Unlike P135gag-myb-ets and the Mr 75,000 translation product of c-myb (P75c-myb), which are nuclear proteins, P54c-ets was found to be predominantly cytoplasmic. P54c-ets is expressed at low levels in most cell lines and tissues tested, including bone marrow cells and circulating lymphocytes. P54c-ets, together with a minor but closely related Mr 56,000 protein, was found to be expressed at high levels in chicken thymocytes and bursal lymphocytes.

The transforming sequences of the avian acute leukemia virus, E26, contain two distinct oncogenes, v-mybE and v-ets, fused together. By using a probe containing v-ets sequences, polyadenylated transcripts of the c-ets proto-oncogene were detected in avian tissues; they included a major 7.0-kilobase and a minor 2.0-kilobase species. These c-ets mRNAs were detected at high levels only in lymphoid organs and in avian T and B lymphoid cell lines. A similar pattern of c-ets transcription was observed in human hematopoietic cell lines, with transcripts detected in lymphoid B and T cells but not in erythroid or myeloid cells. The E26 oncogene was inserted into an inducible expression vector, and a 90-kilodalton protein (bp90) was produced in bacteria. Rabbit antisera raised to purified bp90 precipitated P135gag-mybE-ets, the v-mybE-ets polyprotein expressed in E26-transformed cells, and also reacted with p50v-mybA, the transforming protein of the avian myeloblastosis virus. Antiserum to bp90 was absorbed with a bacterially synthesized v-mybA protein to remove anti-myb activity. The absorbed anti-bp90 serum retained the ability to immunoprecipitate P135gag-mybE-ets from E26-transformed cells and specifically reacted with a 56-kilodalton polypeptide (p56) detected in chicken lymphoid organs and in T and B lymphocytes of both avian and human origin. The data suggest that p56 is a translational product of the c-ets proto-oncogene and imply that p56 may be involved in regulating the growth of lymphoid cells.

Antibodies were prepared against bacterially expressed polypeptides corresponding to various portions of the v-ets-encoded domain of P135gag-myb-ets, the transforming protein of avian leukemia virus E26. Immunoprecipitation analyses show that ca. 80 v-ets-encoded amino-acids located immediately after the v-myb/v-ets junction are not found in P54/56c-ets, the translation product of the c-ets proto-oncogene, nor in a set of cellular proteins of 64, 62, and 60 kDa related to but distinct from P54/56c-ets. In addition, Northern blot analyses show that these 5 v-ets sequences neither derive from the nontranslated region of the known cellular transcripts hybridizing to a v-ets probe nor from the c-myb transcript or the helper virus genetic information. Tryptic peptide analyses furthermore indicate that, except for these sequences and the last 16 carboxy terminal amino acids of P135gag-myb-ets, the amino acids encoded by v-ets are essentially colinear with those of P54c-ets.

We have investigated the structure of chicken genomic DNA homologous to v-ets, the second cell-derived oncogene of avian retrovirus E26. We isolated a c-ets locus spanning ca. 30.0 kilobase pairs (kbp) in the chicken genome with homologies to 1,202 nucleotides (nt) of v-ets (total length, 1,508 nt) distributed in six clusters along 18.0 kbp of the cloned DNA. The 5 -distal part of v-ets (224 nt) was homologous to chicken cellular sequences contained upstream within a single 16.0-kbp EcoRI fragment as two typical exons but not found transcribed into the major 7.5-kb c-ets (or 4.0-kb c-myb) RNA species. Between these two v-ets-related cellular sequences we found ca 40.0 kbp of v-ets-unrelated DNA. Finally, the most 3 region of homology to v-ets in the cloned DNA was shown to consist of a truncated exon lacking the nucleotides coding for the 16 carboxy-terminal amino acids of the viral protein but colinear to one of the two human c-ets loci, c-ets-2.

The avian acute leukemia virus E26 induces erythroblastosis and myeloblastosis in chickens. The oncogene of this virus includes sequences derived from the cellular gene designated c-ets, which is normally expressed in lymphoid cells and whose product is a protein of apparent molecular weight ca. 54,000 daltons. Complementary DNA clones representing the major transcript of the chicken c-ets proto-oncogene were isolated from a spleen cell library. Sequence analysis of the cDNA revealed that it contains an open reading frame encoding a polypeptide of 441 amino acids with a molecular weight of 49,932 daltons. This open reading frame can be transcribed and translated in vitro into a 50 kd protein that is specifically immunoprecipitated with antiserum to the v-ets oncogene product. Within the central region of homology between c-ets and v-ets, there are only 5 nucleotide substitutions resulting in 4 amino acid changes. However, coding sequences at the 5 and 3 ends of the v-ets oncogene and the chicken c-ets cDNA differ from one another. These changes may be responsible for the differential functions of c-ets and v-ets in cells of different hematopoietic lineages and may account for the pathogenic properties of the v-ets oncogene.

The avian acute leukemia virus E26 induces a mixed erythroid-myeloid leukemia in chicken and carries two distinct oncogenes, v-myb and v-ets. It was shown that there exist two ets loci in humans, c-ets-1 and c-ets-2, which were mapped to human chromosome 11 and 21, respectively. The human c-ets-1 proto-oncogene locus was shown to translocate from chromosome 11 to 9 or 4 in certain patients with acute monocytic leukemia. In order to understand the functional role of c-ets-1 proto-oncogene, we have made a cDNA library from MOLT-4 cells and isolated seven c-ets-1 positive cDNA clones. Analysis of two cDNA clones revealed a complete open reading frame of the c-ets-1 proto-oncogene. Except for 81 nucleotides at the 5 -end (downstream of initiation codon) and 39 nucleotides at the 3 -end (upstream of termination codon), the c-ets-1 cDNA is approximately 82% homologous to v-ets oncogene sequences and encodes a 441-residue protein (c-ets-1a) which contains contiguous 5 and 3 regions of the v-ets oncogene as in the case of chicken. Sequence analysis also revealed alternative splicing, giving rise to another related c-ets-1 polypeptide (c-ets-1b) lacking 87 amino acids. Our results suggest that differential RNA splicing allows the synthesis of at least two related c-ets-1 polypeptides from a single c-ets-1 proto-oncogene.

The chromosome localization of a 5.4 kb DNA genomic probe of proto-oncogene c-ets 1 has been analysed in an acute monocytic leukemia with t (1; 11) (q21; q23) translocation. The c-ets probe has been translocated onto the rearranged chromosome 1, suggesting the involvement of the proto-oncogene in leukemias with chromosome rearrangements at band 11 q23.

The v-myb, ets-containing avian leukemia virus E26 is unique in its capacity to transform both erythroblasts and myeloblasts. Previous studies showing that v-myb is sufficient for the transformation of myeloid cells failed to definitively establish the role of the v-ets gene. We have now isolated a mutant of E26, ts1.1, that is temperature-sensitive for erythroid cell transformation and that we found to contain a single mutation in the v-ets gene. Surprisingly, myeloid cells transformed by this mutant showed an altered phenotype relative to wild-type-transformed cells, in that they resemble promyelocytes. In addition, infection of mature macrophages with ts1.1 led to their transformation and conversion into promyelocyte-like cells. We conclude that the v-ets domain of the p135gag-myb-ets protein of E26 has an effect on both erythroid and myeloid cell differentiation, suggesting a possible role for the c-ets/c-myb genes in the commitment of hematopoietic cells towards specific lineages.

The locus SU(Lv)-ets-2 of the sea urchin Lytechinas variegatus related to the oncogene v-ets of avian erythroblastosis virus E26 has been molecularly cloned. The cloned DNA was found to contain a region with a high degree of homology to E26 v-ets. The sea urchin homology with v-ets starts at a consensus splice acceptor sequence and stops at the point where homology between v-ets and human c-ets ends. This region corresponds to the Hu-ets-2 homologous sequences defined by Watson et al. (1985, Proc. Natl. Acad. Sci, USA 82, 7294-7298). Ninety-one out of 97 (or 94%) predicted amino acids are identical between sea urchin c-ets and E26 v-ets over the region of homology. This degree of homology exceeds the maximum homology previously found between any oncogene and an invertebrate homolog. A somewhat weaker homology with the Hu-ets-2 sequences continues beyond, for 13 codons, ending at a common termination codon. Northern blot analysis of mature unfertilized eggs and early embryos from sea urchins of the species Strongylocentrotus purpuratus revealed a single 6.8-kb ets-related RNA that is expressed at a maximum level during the early stages of embryonic development. This RNA species is polyadenylated indicating that it is the message for the sea urchin ets-2 gene.

The avian erythroblastosis virus, E26, an acute leukemia virus, contains a transforming gene composed of two cellular components, v-myb and v-ets. The v-ets related sequences of man and other mammals consist of two transcriptionally active genes, ets-1 and ets-2, located on separate chromosomes. By contrast, both of these genes are contiguous in birds, are located on the same chromosome, and are coordinately transcribed. The human ets-1 and ets-2 gene products were identified by means of antibodies directed against the ets-1 and ets-2 encoded products. A 51 kD protein has been identified as the ets-1 gene product, and a 56 kD protein as the ets-2 gene product. Cellular fractionation studies indicated that the ets-1 protein is located in the cytoplasm and the ets-2 protein is nuclear. By comparison, the chicken ets protein, which contains both the ets-1 and ets-2 domains, distributes equally between the cytoplasm and nucleus. The differential compartmentalization of the ets gene products and their non-coordinate expression suggest that these proteins have different biological functions.

In chicken cells, we previously identified a set of proteins (p58-64) structurally related to, but distinct from, the products encoded by the c-ets proto-oncogene. We report here the isolation and nucleotide sequence of a cDNA encoding nuclear products of mol. wt 58, 60, 62 and 64 kd, indistinguishable from those detected in chicken cells. The p60 and p64 species appear to represent phosphorylated versions on serine and threonine residues of p58 and p62. The homology of p58-64 to other ets-related proteins, including the v-ets encoded domain of the transforming protein of avian leukemia virus E26 and p54c-ets, the translation product of the chicken (Ck) c-ets gene, is confined to two regions of 175 and 96 amino acid residues localized respectively at the carboxy-terminal domain and close to the amino-terminal domain of these molecules. This cDNA corresponds to a gene localized in a locus distinct from that of c-ets which is transcribed as a 4.0-kb RNA species in most chicken tissues. We also identified the human (Hu) c-ets-2-encoded products as two proteins of 60 and 62 kd, highly related to chicken p58-64. This, together with the fact that the amino acid sequence of the cDNA encoding p58-64 is 95% identical to the reported partial sequence of a Hu-c-ets-2 cDNA, indicates that p58-64 are the translation products of the Ck-c-ets-2 gene.

A patient with Ph-negative chronic myeloid leukemia showed active karyotypic evolution when he entered blast crisis. One cell line, which predominated briefly in an accelerated myeloid phase, was characterized by the t(11;19)(q23;p13). Chromosome in situ hybridization demonstrated movement of the oncogene c-ets-1 from the der (11q-) to the der (19p+). The breakpoint at 19p13 was in the vicinity of the human insulin receptor gene locus (INSR). No rearrangements of the c-ets and INSR genes were found in Southern blot analyses. Myeloid lineage was indicated by cell morphology and absence of immunoglobulin JH gene rearrangement and was supported by loss of the germ line bcr-3 gene. Chromosome rearrangements involving 11q23 and movement of c-ets-1 characterize monocytic and lymphoid leukemias and have not previously been reported in myeloid blast crisis of chronic myeloid leukemia.

The synthetic oligonucleotide probes were used for the analysis of N-ras oncogenes detected in human acute leukemias. The mutations of N-ras genes were observed to occur randomly among the subtypes of myeloid leukemias, whereas the N-ras mutations at codon 12 are more likely to occur in lymphoid leukemias than other mutations. The mutations at codon 13 of the N-ras gene were not detected in acute leukemias although they were found in myelodysplastic syndrome that is considered to be a preleukemic state.

We report here significant primary sequence homology among the predicted translational products of three genes: CDC4 , CDC36 and ets. CDC4 and CDC36 are Saccharomyces cerevisiae cell division cycle genes, while ets is a transformation-specific sequence of avian erythroblastosis virus E26. The deduced primary structures of the three gene products were compared by computer to a large data base of known and predicted protein sequences. The search revealed 22.0-25.5% identity over regions of 140-206 codons, respectively between the different pairwise combinations. For these particular sequences, these identity scores fall 3.4-4.0 standard deviations above the empirically-determined mean values of fortuitous similarity. S. cerevisiae calls require CDC36 and CDC4 in order to complete two early events in the cell cycle: execution of start ( CDC36 ) and spindle pole body separation ( CDC4 ). In virus E26, the ets sequence is linked in frame with delta gag and mybE in the tripartite structure 5 -delta gag- mybE -ets-3 , comprising the E26 transforming oncogene. The homologies described here suggest that the biochemical functions or regulation of the CDC4 , CDC36 and ets products may be related.

E26 is an acute leukaemia avian retrovirus which induces myeloblastosis and erythroblastosis in vivo and transforms erythroblasts and myeloblasts in vitro. It contains the oncogene v-myb (ref. 4), first described for avian myeloblastosis virus (AMV), as well as a second specific nucleotide sequence, v-ets located 3 to v-myb (refs 5,6). We have reported that v-ets has a cellular counterpart (c-ets) in chicken and human DNA. Now, using two independent methods--hybridization with human c-ets probe of sorted chromosomes and in situ hybridization--we report the localization of the ets locus on human chromosome 11 at bands q23-q24. This finding may be important, as specific breakpoints around this position have been reported for human malignancies such as acute monocytic leukaemia and Ewing s sarcoma.

The formation of new blood vessels is an essential process in embryonic development and wound healing, for tumor growth and metastasis. In situ hybridization studies have revealed that the protooncogene c-est1 is expressed in endothelial cells at the beginning of blood vessel formation, in normal and pathological conditions. c-ets1 encodes a transcription factor, a protein which binds specifically to DNA and which regulates the transcription of genes containing these specific binding sequences in their promotors. Thus, in vitro experiments suggest that c-ets1 may activate the transcription of genes encoding collagenase 1, stromelysine 1 and urokinase plasminogen activator, proteases involved in extracellular matrix degradation. A working hypothesis is that c-ets1 takes part in regulating angiogenesis by controlling the transcription of these genes whose activity is necessary for the migration of endothelial cells from pre-existing capillaries. This hypothesis is discussed with respect to current experimental evidence and to the complexity of the regulatory network controlling gene transcription and extracellular matrix degradation.

The Ets-1 proto-oncogene is a member of a transcription factor family characterized by homology to the v-ets oncogene. In adult mice, Ets-1 is expressed predominantly in lymphoid cells where it has been implicated in regulating transcription of lymphocyte-specific genes. Following T-cell activation, the specific DNA binding activity of Ets-1 is inactivated by transient phosphorylation, suggesting a function in the transition from the resting to activated state. Ets-1 has also been suggested to cooperate with the AP-1 transcription factor complex to mediate cellular growth factor responses. Here we show, by using RAG-2-deficient blastocyst complementation, that Ets-1 deficiency has dramatic, but different, effects on development and function of T- and B-lineage cells. Ets-1-deficient T cells were present in reduced numbers and were highly susceptible to cell death in vitro. In contrast, Ets-1-deficient B cells were present in normal numbers but a large proportion were IgM plasma cells. Our data demonstrate that Ets-1 is essential for maintenance of the normal pool of resting T- and B-lineage cells.

Apoptosis or programmed cell death represents a mechanism by which cells possessing DNA damage can be deleted. The bcl-2 proto-oncogene is a known inhibitor of apoptosis that may allow the accumulation and propagation of cells containing genetic alterations. To determine if and when the bcl-2 gene is activated during colorectal tumorigenesis and its relationship to p53, we analyzed normal mucosa, hyperplastic and dysplastic epithelial polyps, and carcinomas for the expression of these markers using immunohistochemistry. Whereas bcl-2 staining was restricted to basal epithelial cells in normal and hyperplastic mucosa, bcl-2 expression was detected in parabasal and superficial regions in dysplastic polyps and carcinomas. An inverse correlation was found between bcl-2 and p53 expression in adenomas, suggesting that these markers may regulate a common cell death pathway. Furthermore, carcinomas with a high percentage of bcl-2-positive cells were significantly more likely to have low rates of spontaneous apoptosis, as determined histologically, than those cancers with low or absent bcl-2 expression. Abnormal activation of the bcl-2 gene appears to be an early event in colorectal tumorigenesis that can inhibit apoptosis in vivo and may facilitate tumor progression.

We studied the frequency of a SstI polymorphism of the Ets-1 oncogene in 100 patients with non-Hodgkin s lymphoma, 44 patients with Hodgkin s disease, 49 patients with chronic myeloid leukemia, and 100 controls. There was no difference in the genotype frequency between the controls and patients with either Hodgkin s disease or chronic myeloid leukemia. In contrast, there was a highly significant difference in the distribution of the three genotypes between the patients with non-Hodgkin s lymphoma and the controls (X2 = 10.76, 2df, p = 0.004) with the C2 allele being more frequent in the lymphoma patients. Molecular cloning indicated that the polymorphic SstI site lay 304 bp from exon 7. This is the second association of the SstI polymorphism of the Ets-1 oncogene with a lymphoid disorder and suggests that the presence of the C2 allele is associated with a predisposition to develop a lymphoid malignancy.

The ets gene family encodes a group of proteins which function as transcription factors under physiological conditions and, if aberrantly expressed, can cause cellular transformation. We have recently identified two regulatory elements in the murine immunoglobulin heavy-chain (IgH) enhancer, pi and microB, which exhibit striking similarity to binding sites for ets-related proteins. To identify ets-related transcriptional regulators expressed in pre-B lymphocytes that may interact with either the pi or the microB site, we have used a PCR approach with degenerate oligonucleotides encoding conserved sequences in ALL members of the ets family. We have cloned the gene for a new ets-related transcription factor, ERP (ets-related protein), from the murine pre-B cell line BASC 6C2 and from mouse lung tissue. The ERP protein contains a region of high homology with the ETS DNA-binding domain common to ALL members of the ets transcription factor/oncoprotein family. Three additional smaller regions show homology to the ELK-1 and SAP-1 genes, a subgroup of the ets gene family that interacts with the serum response factor. Full-length ERP expresses only negligible DNA-binding activity by itself. Removal of the carboxy terminus enables ERP to interact with a variety of ets-binding sites including the E74 site, the IgH enhancer pi site, and the lck promoter ets site, suggesting a carboxy-terminal negative regulatory domain. At least three ERP-related transcripts are expressed in a variety of tissues. However, within the B-cell lineage, ERP is highly expressed primarily at early stages of B-lymphocyte development, and expression declines drastically upon B-cell maturation, correlating with the enhancer activity of the IgH pi site. These data suggest that ERP might play a role in B-cell development and in IgH gene regulation.

The c-ets-1 locus encodes two transcription factors, p54c-ets-1 and p68c-ets-1 that recognize purine-rich motifs. The v-ets oncogene of the avian retrovirus E26 differs from its cellular progenitor p68c-ets-1 by two amino acid substitutions (alanine 285 and isoleucine 445 in c-ets-1 both substituted by valine in v-ets, mutations A and B respectively) and its carboxy-terminal end (mutation C). The B mutation affects a well conserved residue in the carboxy-terminal 85 amino acids, ETS DNA-binding domain. To address the biological relevance of the B mutation found between v-ets and c-ets-1, we have randomly mutagenized isoleucine 445 of p68c-ets-1 by polymerase chain reaction. Using in vitro gel mobility shift assays, we show that this residue is crucial for the binding properties of c-ets-1 since the 12 mutations we have generated at this position, ALL diminish or even abolish the binding, to the optimized Ets-1 binding site (EBS), of 35 kDa proteins corresponding to the 311 carboxy-terminal residues of c-ets-1. Among them, substitutions of isoleucine to glutamic acid, glycine or proline have the highest inhibitory effects. Similar results were obtained when the same mutations were introduced either in full-length p68c-ets-1 protein or into a carboxy-terminal polypeptide of 109 amino acids encompassing the ETS-domain which has previously been shown to display a very high binding activity as compared with the full-length protein. Consistent with the in vitro results, point mutations in p68c-ets-1 that decrease binding activity to EBS abrogate its ability to transactivate reporter plasmids carrying either the TPA oncogene Response Unit of the Polyoma virus enhancer (TORU) or a sequence derived from the HTLV-1 LTR. Furthermore, as this isoleucine residue is rather well-conserved within the ETS gene family, we show that mutation of the corresponding isoleucine of c-ets-2 into glycine also abrogates its DNA-binding and hence, transactivating properties. Thus, the v-ets B mutation highlights the isoleucine 445 as an essential amino acid of the c-ets-1 and c-ets-2 DNA-binding domains.

The oncogene v-ets and the proto-oncogene c-ets-1 code for members of the Ets family of transcription factors that bind to DNA motifs comprising GGA(A/T) through the conserved Ets domain. c-Ets-1 and v-Ets are very similar, differing in sequence only at three positions. In this report we demonstrate that v-Ets has a less stringent target sequence requirement than c-Ets-1. v-Ets binds strongly to a broad spectrum of DNA sequence motifs, and consequently, weaker binding sites have a much greater affinity for v-Ets than c-Ets-1. c-Ets-1 carries two inhibitory domains: the D domain present N-terminal to the DNA binding domain and the C-terminal domain which is mutated in v-Ets. Our results show that the D domain has a stronger inhibitory effect than the C-terminal sequence. The on- and off-rates of c-Ets-1 vary greatly depending on the DNA binding sequences, in contrast to those of v-Ets. The c-Ets-1 on- and off-rates are higher with a strong site than with a weak site. Our data suggest that DNA sequences help c-Ets-1 change from a closed to an open, DNA-binding-competent structure, facilitating its binding to DNA, whereas v-Ets functions without such a process presumably because its different C-terminal sequence generates a constitutively open conformation. The loss of a stringent target sequence selectivity by v-Ets suggests that it might transform cells by altering expression of tightly regulated genes with non-consensus Ets binding sites.

V-ets was found as an oncogenic sequence in the E26 avian retrovirus, which produced leukemias in chickens. There are a large number of mammalian homologues of v-ets, ALL sharing a common sequence called the ETS domain. These mammalian homologues have been found to be transcriptional activators, and the ETS domain is the DNA binding portion of the gene. This family has been found to be important in regulating embryonic development and the response to growth stimuli. In addition, PU.1 and FLI-1 are dysregulated by Friend leukemia virus insertion in mice resulting in erythroleukemia. Significantly, the DNA binding portion of FLI-1 is the 3 part of an oncogenic fusion transcript (termed EWS-FLI) in human Ewing s sarcoma and neuroepithelioma.

Many of the Ets proteins have been shown to be transcription activators. In vitro, Ets 1 proteins are involved in the transcriptional induction of genes such as stromelysin 1, collagenase 1 or urokinase type plasminogen activator, which are proteases responsible for extracellular matrix degradation. In vivo, c-ets 1 is expressed in a wide variety of embryonic tissues in migrating cells, especially in endothelial cells during blood vessel formation. C-ets 1 is also expressed in stromal cells of invasive carcinomas. In the present work, we have investigated the expression of both c-ets 1 and u-PA, a putative target gene of the Ets 1 proteins, within a biological model which includes both embryonic and tumoral aspects. Implantation and placentation of the mouse embryo display migration of the trophoblastic cells, which invade the stroma of the uterine endometrium and trigger the establishment of a new vascular frame. Using in situ hybridization, we show that the overlapping of expression of c-ets 1 and u-PA is restricted to some maternal cell populations from the invasive front and to the endothelial cells of the endometrial vasculature. C-ets 1 is never expressed in trophoblasts. In contrast, u-PA expression in trophoblasts is strong and coincides with the embryo invasive phase. In the embryo proper, c-ets 1 displays a spatio-temporal expression pattern similar to that described in the chick embryo. Until E 10.5, u-PA is expressed neither in embryonic nor in extra-embryonic structures. The respective roles of c-ets 1 and u-PA and their relationship during mammalian placentation are discussed.

Members of the ets gene family encode transcription factors that regulate the expression of a variety of cellular and viral genes including several protooncogenes. We have utilized Drosophila to elucidate the in vivo function of one family member. We show by complementation rescue and sequence analysis that the female sterile mutant tiny eggs (tne) is an allele of the Drosophila Ets-related gene Elg (also called D-elg). The mutation of a highly conserved tyrosine residue in the ETS DNA-binding domain of the Elg gene product demonstrates that normal gene function is required for proper follicle cell migration, chorion formation, and nurse cell-chromosome decondensation during Drosophila oogenesis.

We have used genetic complementation rescue to identify two lethal alleles of D-elg, an ets proto-oncogene related gene of Drosophila. Animals that are hemizygous or trans-heterozygous for the alleles die as pharate adults, demonstrating normal gene function is required to complete Drosophila development. Females trans-heterozygous for the 1(3)902 lethal allele and the previously characterized tne female sterile allele produce embryos with abdominal segmentation defects. This finding implicates a role for the D-elg gene in anterior-posterior patterning. The cloning and sequencing of the lethal alleles identified molecular mutations that may result in ELG protein truncation, altered ELG protein interactions, or defective D-elg mRNA splicing.

The segment of the avian leukemia virus E26 genome near the termination of the p135gag-myb-ets open reading frame contains an inversion of the chicken ets-1 sequence. The inversion contains at least 41 bp and may be as large as 46 bp. This results in the replacement of 13 amino acids of chicken ets-1, with 16 amino acids derived from reverse complement of the normal ets-1 coding strand or read-through into E26 env sequences. At least 13 of these codons are specified by the inverted ets sequences. This represents the first reported occurrence of inverted oncogene sequences in a natural retrovirus. The inverted ets sequences are immediately followed by sequences homologous to the Rous sarcoma virus Prague B env gene. Since the E26 env sequence is more closely related to subgroup B avian retroviruses than to avian retroviruses from subgroups A, C, D, or E, the progenitor of E26 was a virus belonging to avian retrovirus subgroup B.

The v-ets-encoded domain in the P135gag-myb-ets transforming protein of the E26 retrovirus differs mainly from its cellular progenitor, p68c-ets-1 by two point mutations and by the replacement of the 13 last C-terminal amino acids present in c-ets-1 by 16 unrelated residues of previously unknown origin in v-ets. Here, we demonstrate that these v-ets C-terminal specific residues are in fact encoded by the opposite strand of the c-ets-1 C-terminus.

6-Phosphofructo-2-kinase (EC 2.7.1.105)/fructose-2,6-bis-phosphatase (EC 3.1.3.46) catalyzes the synthesis and degradation of fructose 2,6-bisphosphate, a ubiquitous stimulator of glycolysis. The liver (L-type) and muscle (M-type) mRNAs for this bifunctional enzyme arise from distinct promoters of the same gene. We have now characterized in rat hepatoma FTO2B cells another mRNA, which is transcribed from a third promoter of that gene. This F-type mRNA is present in fetal rat liver and muscle, in rat placenta, and in several established rat cell lines. The F promoter contains no TATA box but contains several binding sites for Sp1 and for members of the ets oncogene family. Transfection of FTO2B cells with constructs containing the intact or mutagenized F promoter showed that its activity depends mainly on one of these sites. This site bound a heteromeric FTO2B cell protein indistinguishable from the ets-related GA binding protein alpha/ankyrin-repeats GA binding protein beta transcription factor.

The avian acute leukemia virus E26 encodes a fusion protein between viral Gag and the cellular transcription factors cMyb and cEts1(p68). vEts on its own transforms more mature erythroid cells. We have compared the properties of vEts and cEts1(p68). vEts interacts preferentially with an antibody that recognizes the active conformation of the DNA-binding domain. The DNA-binding activity of vEts is particularly sensitive to incubation conditions for band-shift assays, phosphorylation and modification by sulphydryl-specific reagents. Increased sensitivity is due to loss of a protective function of cEts1 C-terminal sequences. cEts2 has a related C-terminal sequence with a similar role. These results suggest that the vEts DNA-binding domain is more accessible to protein-protein interactions and to regulatory mechanisms. Indeed, vEts DNA binding is preferentially inactivated by oxidizing conditions in-vivo. We suggest that the open conformation of the vEts DNA-binding domain favours interactions with other proteins or DNA and facilitates transformation.

The recently cloned fli-1 gene is a member of the ets oncogene family that is preferentially expressed in hematopoietic cells. It is a target of dysregulation by Friend leukemia virus insertion and translocation in Ewing s sarcoma and neuroepithelioma. In this report, we have studied the function and regulation of both murine and human fli-1. Analysis of the human and mouse fli-1 proteins showed that fli-1 binds to specific DNA sequences highly related to m-ets-2 binding sites. Methylation protection experiments showed that fli-1 and m-ets-2 contacted the same nucleotides in two different binding sites. The fli-1 protein was shown to be a transcriptional activator in co-transfection studies. Stimulation of murine bone marrow macrophages by mediators of inflammation, such as lipopolysaccharide, phorbol 12-myristate 13-acetate, interleukin-1, and interferon-gamma resulted in the reduced expression of fli-1 mRNA. fli-1 was only expressed in a defined subset of human erythroleukemia cell lines.

In various invasive human tumors, c-ets-1 mRNA was found to be selectively expressed in stromal fibroblasts. We have now investigated the possibility that soluble factors could regulate c-ets-1 expression in cultured human fibroblasts. We show that both conditioned media from tumor cell lines and a number of characterized cytokines and growth factors were able to induce c-ets-1 expression. TNF alpha and IL-1 alpha were the most potent c-ets-1 stimulators, inducing rapid (within 1 h) and long-lasting (19 h) increases of c-ets-1 mRNA and protein expression. In contrast, bFGF, EGF, and PDGF were mainly delayed stimulators, with maximal stimulation being detected by 19 h. In addition, these growth factors potentiated the rapid induction of c-ets-1 by TNF alpha. While ALL these factors were able to stimulate c-ets-1 expression, TGF beta was found to be ineffective. Using inhibitors of transcription and translation, we also found that increase of c-ets-1 mRNA by TNF alpha resulted from new transcription rather than from stabilization and did not require new protein synthesis. These results demonstrated that c-ets-1 is a new nuclear target for several factors and behaves as an early-response gene for TNF alpha.

The P4 promoter of parvovirus minute virus of mice (MVMp) directs transcription of the genes coding for nonstructural proteins. The activity of promoter P4 is regulated by several cis-acting DNA elements. Among these, a promoter-proximal GC box was shown to be essential for P4 activity (J.K. Ahn, B.J. Gavin, G. Kumar, and D.C. Ward, J. Virol. 63:5425-5439, 1989). In this study, a motif homologous to an Ets transcription factor-binding site (EBS), located immediately upstream from the GC box, was found to be required for the full activity of promoter P4 in the ras-transformed rat fibroblast cell line FREJ4. In normal parental FR3T3 cells, the transcriptional function of P4 EBS was insignificant but could be restored by transient cell transfection with the c-Ha-ras oncogene. P4 EBS may thus contribute to the stimulation of promoter P4 in ras-transformed cells. Electrophoretic mobility shift assays using crude extracts from FREJ4 cells revealed the binding of a member(s) of the Ets family of transcription factors to the P4 EBS, as well as the interaction of two members of the Sp1 family, Sp1 and Sp3, with the adjacent GC box. When produced in Drosophila melanogaster SL2 cells, Ets-1 and Sp1 proteins acted synergistically to transactivate promoter P4 through their respective cognate sites.

Does the c-ets 1 protooncogene take part in the regulation of tumor angiogenesis? The formation of new blood vessels is an essential process in embryonic development and wound healing, for tumor growth and metastasis. In situ hybridization studies have revealed that the protooncogene c-ets 1 is expressed in endothelial cells at the beginning of blood vessel formation, in normal and pathological conditions. C-ets 1 encodes a transcription factor, a protein which binds specifically to DNA and which regulates the transcription of genes containing these specific binding sequences in their promotors. Thus in vitro experiments suggest that c-ets 1 may activate the transcription of genes encoding collagenase 1, stromelysine 1 and urokinase plasminogen activator, proteases involved in extracellular matrix degradation. A working hypothesis is that c-ets 1 takes part in regulating angiogenesis by controlling the transcription of these genes whose activity is necessary for the migration of endothelial cells from preexisting capillaries. This hypothesis is discussed with respect to current experimental evidences and to the complexity of the regulatory network controlling gene transcription and extracellular matrix degradation.

We studied the frequency of an Sst I polymorphism of the ETS-I oncogene in 122 elderly subjects (mean age 78.14 years) and 115 teenagers (mean age 16.9 years). No difference in the frequency of the three genotypes (C1C1, C1C2, C2C2) was found between the two groups. However, the C2 allele occurred more frequently in the elderly subjects (chi 2 = 5.49, P < 0.02). These data suggest that the presence of the C2 allele may be associated with survival to old age.

The paired box transcription factor Pax-5 (B-cell-specific activator protein) is a key regulator of lineage-specific gene expression and differentiation in B-lymphocytes. We show that Pax-5 functions as a cell type-specific docking protein that facilitates binding of the early B-cell-specific mb-1 promoter by proteins of the Ets proto-oncogene family. Transcriptional activity of the mb-1 promoter in pre-B-cells is critically dependent on binding sites for Pax-5:Ets complexes. Ternary complex assembly requires only the Pax-5 paired box and ETS DNA-binding domains. mutation of a single base pair in the ternary complex binding site allows for independent binding by Ets proteins but, conversely, inhibits the binding of Pax-5 by itself. Strikingly, the mutation reverses the pattern of complex assembly: Ets proteins recruit Pax-5 to bind the mutated sequence. Recruitment of Net and Elk-1, but not SAP1a, by Pax-5 defines a functional difference between closely related Ets proteins. Replacement of a valine (V68) in the ETS domain of SAP1a by aspartic acid (as found in c-Ets-1, Elk-1, and Net) enhanced ternary complex formation by more than 60-fold. Together, these observations define novel transcription factor interactions that regulate gene expression in B cells.

The proto-oncogene Ets-1 is a transcription factor known to control the expression of a number of genes involved in extracellular matrix remodeling and has been postulated to play a role in cell migration and tumor invasion. To elucidate the involvement of Ets-1 in human gastric carcinomas, we examined 11 cases of gastric adenoma and 110 cases of gastric carcinoma by immunohistochemistry and compared the degree of Ets-1 expression with the depth of carcinoma invasion. Ets-1 was not expressed either in the normal gastric epithelium or in gastric adenomas. Among the 110 cases with gastric adenocarcinoma, 70 (63.6%) showed positive staining for the Ets-1 protein. In mucosal carcinomas, only 3 of 26 cases (11.5%) showed positive immunostaining for Ets-1. In contrast, 67 of 84 cases (79.8%) with submucosal or more invasive carcinomas showed immunopositivity and intense staining for Ets-1 in the tumor cells. The pattern of Ets-1 immunostaining in mucosal carcinomas was weak and differed from that of other local invasive carcinomas (P < 0.001). Histologically, signet-ring cell and mucinous carcinomas expressed relatively weak positivity for Ets-1. Ets-1 expression correlated significantly with the presence of lymph node metastasis (P < 0.001). In situ hybridization, using an Ets-1 oligonucleotide probe, also confirmed the presence of Ets-1 mRNA in gastric carcinomas. expression of Ets-1 mRNA was also detected in four different kinds of cultured human gastric carcinoma cell lines by the reverse transcription polymerase chain reaction method. These findings suggest that Ets-1 is overexpressed in gastric mucosal cells that have undergone malignant conversion and that Ets-1 is one of the factors involved in the penetration of gastric carcinoma beyond the muscularis mucosa.

TEL is a novel member of the ETS family of transcriptional regulators which is frequently involved in human leukemias as the result of specific chromosomal translocations. We show here by co-immunoprecipitation and GST chromatography analyses that TEL and TEL-derived fusion proteins form homotypic oligomers in vitro and in vivo. Deletion mutagenesis identifies the TEL oligomerization domain as a 65 amino acid region which is conserved in a subset of the ETS proteins including ETS-1, ETS-2, FLI-1, ERG-2 and GABP alpha in vertebrates and PNTP2, YAN and ELG in Drosophila. TEL-induced oligomerization is shown to be essential for the constitutive activation of the protein kinase activity and mitogenic properties of TEL-platelet derived growth factor receptor beta (PDGFR beta), a fusion oncoprotein characteristic of the leukemic cells of chronic myelomonocytic leukemia harboring a t(5;12) chromosomal translocation. Swapping experiments in which the TEL oligomerization domain was exchanged by the homologous domains of representative vertebrate ETS proteins including ETS-1, ERG-2 and GABP alpha show that oligomerization is a specific property of the TEL amino-terminal conserved domain. These results indicate that the amino-terminal domain conserved in a subset of the ETS proteins has evolved to generate a specialized protein-protein interaction interface which is likely to be an important determinant of their specificity as transcriptional regulators.

p21(Waf1/Cip1) is one of the key regulatory proteins in cell cycle, terminal differentiation, and apoptosis. Its promoter was shown to be transactivated by the wild-type p53 protein as well as in a p53-independent manner. In this report, we demonstrate that E1AF, an ets-related transcription factor, activates the human p21(Waf1/Cip1) promoter by interacting with the ets-binding sites located close to the two previously identified p53-responsive elements. Northern blot analysis revealed that p21(Waf1/Cip1) and E1AF were correlatively upregulated in response to cisplatin treatment in SiHa cells. Transient expression assays demonstrated that E1AF can activate the p21(Waf1/Cip1) promoter-driven luciferase reporter gene in SiHa cells. The p21(Waf1/Cip1) promoter activity was also increased in p53-null Saos2 osteosarcoma cells, but was markedly reduced when the ets-binding sites were deleted. These results indicate that E1AF positively regulates transcription from the p21(Waf1/Cip1) promoter in response to genotoxic stresses.

First member of Ets gene family was discovered a decade ago by studying avian erythroblastosis virus, E26. This virus encodes a tripartite protein gag-myb-ets with a molecular weight of 135 kDa. Subsequently, a series of cellular Ets genes were isolated (Ets-1, Ets-2, Erg, Elk-1, Sap-1, PEA-3, PU.1, Fli-1, Pok/Yan, Etv-1 etc.). These genes share sequence homology to E26 Ets gene (v-ets or viral ets). Ets genes are highly conserved in phylogenetically divergent species from Drosophila to man. Mammalian Ets genes are located on different chromosomes. Ets gene products are transcriptionally active sequence-specific DNA binding proteins and are differentially regulated. Ets genes are involved in certain chromosomal translocations leading to the formation of chimeric fusion proteins that are associated with certain leukemias and soft tissue cancers. Ets genes also have a role in T-cell development and molecular and genetic analysis of Down Syndrome patients have implicated the human Ets-2 and Erg genes in the disease. Down Syndrome afflicted patients have immunologic and thymic disorders as well as a greater risk for leukemic disease. Thus, Ets genes having homology to viral oncogenes, may be instrumental in regulating cellular growth and differentiation, as well as organismal development. Alteration of these genes and their products may cause deregulation of normal cell growth and differentiation and result in a disease.

The proto-oncogene, ets-1, is a transcription factor that controls the expression of a number of genes involved in extracellular matrix remodeling. It might play a role in cell migration and tumor invasion. To elucidate the involvement of Ets-1 in human pancreatic carcinoma, we performed immunohistochemical analysis on tissue from 10 normal pancreases and 103 cases of pancreatic carcinoma. We compared the degree of Ets-1 expression. In addition, among the pancreatic carcinomas, we compared Ets-1 expression in relation to the differentiation, lymph node metastasis and the depth of invasion of the carcinoma. Ets-1 was expressed faintly in normal pancreatic tissue. Among the 103 cases of pancreatic carcinoma, 83 (80.5%) showed positive staining for the Ets-1 protein. Histologically, papillary carcinoma, well-differentiated adenocarcinoma, and moderately differentiated adenocarcinoma expressed high positivity for Ets-1. In contrast, poorly differentiated adenocarcinoma expressed relatively weak positivity for Ets-1. Ets-1 expression had no relation to the presence of lymph node metastasis, tumor size, prognosis, or tumor-node-metastasis stage in pancreatic carcinomas. In situ hybridization also confirmed the presence of ets-1 mRNA in pancreatic carcinomas. We detected expression of ets-1 mRNA in three human pancreatic carcinoma cell lines by the reverse transcription-polymerase chain reaction method. These findings suggest that Ets-1 expression is related to the carcinogenesis of human pancreatic carcinoma, but its relationship to tumor progression is unclear.

The ets family is a large multigene family of transcription factors that share a conserved DNA-binding "ETS" domain and include several oncoproteins that induce tumorigenesis when overexpressed. Here we report the cDNA cloning from mouse pituitary somatotroph tumors, sequence characterization and tissue-specific expression pattern in mice of a novel ets family gene, "Ehf" ("ets homologous factor"). The putative 300 amino acid Ehf protein is a highly divergent ets family member, but is most related to the recently identified oncoprotein ESX (36% overall and 84% ETS domain amino acid identity). Thus, Ehf and ESX comprise a new ets subfamily. Ehf is a single-copy gene, but produces four distinct mRNA transcripts. Ehf transcripts are abundant in mouse kidney and lung, less so in muscle and liver, and not detected in brain, spleen or testes. Because of its presence in somatotroph tumors and its relationship to ESX, Ehf may represent a new oncoprotein.

The proto-oncogene ets-1 is a transcription factor known to control the expression of a number of genes involved in extracellular matrix remodeling. To elucidate the involvement of ets-1 in human thyroid carcinoma, we examined 68 cases of thyroid carcinoma and 38 cases of benign tumors by immunohistochemical means. ets-1 was not expressed in normal thyroid follicular cells. Among the 69 cases of thyroid carcinoma, 61 (89.7%) showed positive staining for the ets-1 protein. According to histologic classification, 47 (97.9%) of 48 papillary carcinomas, 7 (87.5%) of 8 follicular carcinomas, 3 (60.0%) of 5 medullary carcinomas, and 4 (50.0%) of 8 anaplastic carcinomas showed positive staining of ets-1. Among the benign tumors, staining was positive for ets-1 in 8 (40.0%) of 20 adenomas and 3 (16.7%) of adenomatous goiters. In situ hybridization also confirmed the expression of ets-1 mRNA in thyroid carcinoma. expression of ets-1 mRNA was seen in three kinds of cultured human thyroid carcinoma cell lines and carcinoma tissue obtained at surgery by reverse transcription-polymerase chain reaction. These findings suggest that ets-1 is deeply involved in thyroid carcinogenesis.