Analysis of the expression of cell cycle regulators in Ewing cell lines: EWS-FLI-1 modulates p57KIP2and c-Myc expression.

Ewing tumour is characterized by specific chromosome translocations which fuse EWS to a subset of genes encoding ETS transcription factors, most frequently FLI-1. We report the analysis of the expression of various cell cycle regulators both in Ewing tumour derived cell lines and in different cellular models with either inducible or constitutive EWS-FLI-1 cDNA expression. In Ewing cell lines, cyclin D1, CDK4, Rb, p27KIP1 and c-Myc were consistently highly expressed whereas p57KIP2, p15INK4B and p14ARF demonstrated undetectable or low expression levels. The amount of p16INK4A, p21CIP1, p18INKAC and CDK6 was variable from one cell line to the other. The inducible expression of EWS-FLI-1 led to a strong upregulation of c-Myc and a considerable downregulation of p57KIP2. Other proteins did not show evident modification. High c-Myc and very low p57KIP2 expression levels were also observed in neuroblastoma NGP cells constitutively expressing EWS-FLI-1 as compared to parental cells. Analysis of the p57KIP2 promoter indicated that EWS-FLI-1 downregulates, possibly through an indirect mechanism, the transcription of this gene. Finally, we show that ectopic expression of p57KIP2 in Ewing cells blocks proliferation through a complete G1 arrest. These results suggest that the modulation of p57(KIP2) expression by EWS-FLI-1 is a fundamental step in Ewing tumorigenesis.

Characterization of a new brain-specific isoform of the EWS oncoprotein.

EWS and related TAFII68 and TLS/FUS genes are fused with different genes encoding transcription factors in various human cancers. The products of these genes have the ability to bind RNA and have been shown to be part of splicing and transcription complexes. We show that the EWS, TAFII68 and TLS/FUS proteins are expressed to various levels in all adult murine tissues. We characterize a new isoform of EWS that is specifically expressed in the central nervous system, in both mice and humans. It is shown to be related to a splice variant which includes a new 18-bp exon, termed 4', between exon 4 and 5. The detection of this isoform in spontaneously differentiating SH-SY5Y neuroblastoma cells and in nerve growth factor-induced PC12 cells further links this isoform to neural differentiation. RT-PCR experiments indicate that the level of expression of the brain-specific EWS isoform is stable during brain development whereas that of the ubiquitous EWS isoform decreases during this period. The two isoforms show a parallel decrease in expression after birth. The 4' exon is not detected in tumour-specific EWS fusion transcripts, suggesting that its presence may impair their oncogenic properties. Interestingly, sequences of the 4' exon and flanking regions show remarkable similarities to that of the neural-specific c-src exon, suggesting common mechanisms for the alternative splicing of these exons. The phylogenetic conservation and relationship to neural differentiation strongly suggests an important functional role for this exon.

Cryptic exons as a source of increased diversity of Ewing tumor-associated EWS-FLI1 chimeric products.

In the Ewing family of tumors (EFT), the EWS gene is rearranged with members of the ets oncogene family. Variability in genomic breakpoint locations is the source of significant heterogeneity in fusion product structure. As a consequence of variably included exon sequences from the two partner genes, a variable amount of additional peptide sequence is inserted in between the minimal transforming domains. Some of this molecular diversity has recently been correlated with disparate clinical outcome. Here we report on cryptic exons found in the chimeric RNA of three EFT with different EWS-FLI1 fusions. In two tumors, the emergence of a cryptic exon from FLI1 intron 5 in the chimeric RNA was apparently unrelated to the genomic rearrangement that occurred in FLI1 introns 4 and 5, respectively. In one case, a novel exon was generated through the creation of an artificial splice acceptor site in FLI1 intron 6 by the genomic rearrangement that occurred in EWS intron 8. These results further extend the spectrum of molecular diversity in EFT.

EWS, but not EWS-FLI-1, is associated with both TFIID and RNA polymerase II: interactions between two members of the TET family, EWS and hTAFII68, and subunits of TFIID and RNA polymerase II complexes.

The t(11;22) chromosomal translocation specifically linked to Ewing sarcoma and primitive neuroectodermal tumor results in a chimeric molecule fusing the amino-terminus-encoding region of the EWS gene to the carboxyl-terminal DNA-binding domain encoded by the FLI-1 gene. As the function of the protein encoded by the EWS gene remains unknown, we investigated the putative role of EWS in RNA polymerase II (Pol II) transcription by comparing its activity with that of its structural homolog, hTAFII68. We demonstrate that a portion of EWS is able to associate with the basal transcription factor TFIID, which is composed of the TATA-binding protein (TBP) and TBP-associated factors (TAFIIs). In vitro binding studies revealed that both EWS and hTAFII68 interact with the same TFIID subunits, suggesting that the presence of EWS and that of hTAFII68 in the same TFIID complex may be mutually exclusive. Moreover, EWS is not exclusively associated with TFIID but, similarly to hTAFII68, is also associated with the Pol II complex. The subunits of Pol II that interact with EWS and hTAFII68 have been identified, confirming the association with the polymerase. In contrast to EWS, the tumorigenic EWS-FLI-1 fusion protein is not associated with either TFIID or Pol II in Ewing cell nuclear extracts. These observations suggest that EWS and EWS-FLI-1 may play different roles in Pol II transcription.

Production and characterization of mouse monoclonal antibodies to wild-type and oncogenic FLI-1 proteins.

Mouse monoclonal antibodies were raised against the C-terminal domain of human FLI-1, a member of the ETS family of transcription factors which is involved in various murine and human malignancies. This FLI-1 specific domain is included in the fusion product EWS-FLI-1, an oncogenic variant of FLI-1 expressed in Ewing tumors. Antibodies were screened first by enzyme-linked immunosorbent assay onto recombinant FLI-1-coated plates. Positive clones were then tested for their ability to immunoprecipitate over-expressed EWS-FLI-1 protein. Three monoclonal antibodies were selected and further characterized. One of them, termed 7.3 MoAb, was shown to react with FLI-1 and EWS-FLI-1 in immunoblotting, immunoprecipitation, and immunofluorescence experiments. With all three methods, this antibody not only enabled the detection of overexpressed proteins but also more interestingly, that of endogenously expressed proteins. Furthermore, the 7.3 MoAb can specifically inhibit the interaction of FLI-1 with its DNA-binding site as shown by electrophoretic mobility shift assay. The 7.3 MoAb appears to be specific for FLI-1 because it does not react with ERG, the ETS family member most closely related to FLI-1. This antibody should be a useful tool in the diagnostic evaluation of Ewing tumors and should permit biochemical analyses to study the function of the wild-type FLI-1 protein and of the EWS-FLI-1 fusion protein.

Multiple chromosomal mechanisms generate an EWS/FLI1 or an EWS/ERG fusion gene in Ewing tumors.

The t(11;22)(q24;q12) translocation is found in about 85% of Ewing tumors and leads in all cases to an EWS/FLI1 fusion gene. In a few instances, complex translocations, involving chromosomes 11, 22 and a third chromosome or other variant translocations not involving chromosome 11 also have been reported. These rearrangements generate an active fusion gene between the EWS gene and either the human FLI1 gene or other members of the ETS gene family: the ERG gene localized in band 21q22.2, the ETV1 gene localized in band 7p22, or the E1AF gene localized in band 17q12. Using fluorescence in situ hybridization (FISH) on interphase nuclei or metaphases, we report here the characterization of particular karyotypes in Ewing tumors, namely a complex t(2;11;22) translocation, a variant t(12;22) translocation, and one Ewing tumor without specific rearrangements but with an EWS/ERG fusion gene demonstrated by molecular analysis. These molecular cytogenetic methods allowed us (1) to precisely localize the genomic breakpoints within-EWSR1 and EWSR2 and to identify the chromosome carrying the fusion gene; (2) to determine the nature of events generating the fusion genes; (3) to demonstrate that some variant translocations represent masked complex translocations; and (4) to show that the generation of an EWS/ERG fusion gene cannot be obtained through a simple balanced translocation.

Localization of TEC to 9q22.3-q31 by fluorescence in situ hybridization.

The TEC gene encodes for a novel orphan nuclear receptor. Recently, it has been shown to be involved in the recurrent t(9;22) translocation observed in extraskeletal myxoid chondrosarcoma, in a fusion gene with the EWS gene. We report her on its precise localization on chromosome 9 by fluorescence in situ hybridization.

Structure and promoter characterization of the human stromelysin-3 gene.

In the present study, we have isolated the human stromelysin-3 (ST3) gene which encodes a matrix metalloproteinase (MMP) expressed in fibroblastic cells of tissues associated with intense remodeling. The gene was found to span 11.5 kilobases (kb) including 8 exons and 7 introns. The genomic organization of ST3 gene exons is well conserved compared to other members of the MMP family, except for the 3 last exons corresponding to the hemopexin-like domain and to a long 3'-untranslated region. The transcription initiation site was located 31 nucleotides downstream of a TATA box. Analysis of 1.4 kb of 5'-flanking DNA sequence in the ST3 gene promoter revealed the presence of putative regulatory elements, but no consensus sequence for AP1-binding site in contrast to other MMP promoters. However, a specific cis-acting retinoic acid responsive element of the DR1 type was identified in the proximal region (-385) of the ST3 gene promoter. Transient transfection experiments demonstrated that a minimal promotor activity could be modulated by various sequences within the 3.4 kb of 5'-flanking region, and that the ST3 promoter was transactivated by retinoic acid receptors in the presence of retinoic acid. These findings indicate that the human ST3 gene promoter is characterized by structural and functional features which differ from those previously described in other MMP promoters, and further supports the possibility that ST3 gene expression is controlled by specific factors during tissue remodeling.

Sensitive detection of occult Ewing's cells by the reverse transcriptase-polymerase chain reaction.

Recently, Ewing's tumours have been shown to carry specific hybrid transcripts resulting from the fusion of the EWS gene with FLI-1 or ERG genes. Based on the sensitivity and specificity of the detection of these alterations by the reverse transcriptase-polymerase chain reaction technique, we have developed an assay to search for small numbers of Ewing cells in various sites from patients with Ewing's tumour. This method enables the detection of fewer than one tumour cell per million blood mononuclear cells. A total of 28 primary sites and 51 peripheral samples from 36 patients were investigated. Tumour cells could be detected in 4/18 blood samples, 4/15 bone marrow aspirates and 2/18 peripheral stem cell harvests. EWS/FLI-1 and EWS/ERG transcripts being observed in eight and two cases respectively. The type of fusion transcript detected in peripheral site(s) was identical to that observed in the primary site. At diagnosis 5/16 patients (31%) demonstrated either circulating tumour cells or/and occult bone marrow metastasis. After induction therapy, tumour cells were detected in 3/21 patients. This highly sensitive method should be a relevant tool to allow a more accurate clinical assessment of the dissemination of Ewing's tumours.

High resolution genetic map of the adenomatous polyposis coli gene (APC) region.

Familial adenomatous polyposis coli (APC) is a dominantly inherited colorectal cancer susceptibility disease caused by mutation in a gene called APC and located on chromosome 5q21. Presymptomatic diagnosis of this condition is recommended because it enables restriction of the efficient but demanding prevention program to those relatives that are genetically affected. The large size of the APC gene makes the direct search for the causal alteration difficult to implement in routine diagnostic laboratories. Because APC appears to be genetically homogeneous with alteration in a single locus causing the disease, cosegregation analysis may represent an alternative efficient method for presymptomatic diagnosis. However, the reliability of the risk estimation by linkage analysis in APC families is hampered by the lack of a short range genetic map of the APC locus. A combined approach including genotyping of 65 APC families, analysis of the CEPH database, and complementary typing of both APC and CEPH families has made it possible to derive the following genetic map: Centromere-[D5S82-D5S49]-0.02-D5S122-0.01-D5S136 -0.01-D5S135-0.02-[APC-D5S346-MCC]-0.04-[D5S81-D5S6 4]-Telomere. This order, which differs from previously proposed genetic maps, is fully compatible with recent physical mapping data. These data should contribute to increase the reliability of the presymptomatic test for APC.

The Ewing family of tumors--a subgroup of small-round-cell tumors defined by specific chimeric transcripts.

BACKGROUND: Precise diagnosis of small-round-cell tumors is often a challenge to the pathologist and the clinical oncologist. In Ewing's sarcomas and related peripheral primitive neuroectodermal tumors, a t(11;22) translocation or a (21,22) rearrangement is associated with hybrid transcripts of the EWS gene with the FLI1 or ERG gene. To investigate the diagnostic implication of this observation, we searched for these hybrid transcripts in tumors from patients with clinical and radiologic features of Ewing's sarcoma or peripheral primitive neuroectodermal tumors. METHODS: Samples of RNA from 114 tumors were reverse transcribed and subjected to the polymerase chain reaction with primers designed to amplify the relevant chimeric transcripts. All amplified products were sequenced. RESULTS: In-frame hybrid transcripts were observed in 89 cases. A hybrid transcript was found in 83 of 87 cases (95 percent) of Ewing's sarcoma or peripheral primitive neuroectodermal tumors. Samples of RNA from all of 12 tumors that had been proved to be other than Ewing's sarcoma or neuroectodermal tumors had no hybrid transcript. However, 6 of 15 undifferentiated tumors whose type was ambiguous (nonsecreting, poorly differentiated neuroblastoma or undifferentiated sarcoma) contained a hybrid transcript, suggesting that they might have to be reclassified. CONCLUSIONS: A subgroup of small-round-cell tumors identified as belonging to the Ewing family of tumors can be defined according to a specific molecular genetic lesion that is detectable by a rapid, reliable, and efficient method. This approach can be applied to small specimens obtained by fine-needle biopsies.

Refined mapping of eight cosmid markers on human chromosome 22.

Eight cosmid clones were regionally assigned to small subregions of chromosome 22 by hybridization with a total of 22 somatic cell hybrids. One cosmid was localized to the proximal part of 22q which contained the region commonly deleted in the DiGeorge syndrome. Seven cosmids showing restriction fragment length polymorphisms were localized to the telomeric region distal to the MB locus, which was reported to be frequently deleted in sporadic meningioma. These cosmids, when finely mapped and ordered, are considered useful for the identification of genetic alterations on this chromosome arm.

p53 mutations in human tumors with chimeric EWS/FLI-1 genes.

The Ewing family of tumors is recurrently characterized at the molecular level by the presence of a fusion transcript between the EWS gene on chromosome 22 and either the FLI-I or ERG genes, 2 closely related members of the Ets family of transcription factors. We have investigated 12 primary human tumors, 11 xenografts and 11 cell lines, which have been shown to express chimeric EWS transcripts in search of p53 mutations. Fragments of exons 5 to 8 and the corresponding consensus splice sequences were amplified by PCR and analyzed by denaturing gradient gel electrophoresis (DGGE). In 12 of 34 samples p53 mutations were detected (including 4 samples with multiple p53 mutations). The distribution of the mutations in the various samples was as follows: primary tumors 2/12; cell lines 5/11; xenografts 5/11. No correlation between the presence or absence of p53 mutations and the presence of a specific EWS chimeric transcript was observed. In addition, we observed that p53 mutations were almost always associated with a second hit (either deletion or second mutation) on the other p53 allele.

Interphase molecular cytogenetics of Ewing's sarcoma and peripheral neuroepithelioma t(11;22) with flanking and overlapping cosmid probes.

The translocation, t(11;22)(q24;q12), recurrently observed in Ewing's sarcoma and in peripheral neuroepithelioma has been recently cloned. The analysis of a series of ES/PNE has revealed that the chromosome 22 breakpoints are clustered in a small region of 7 kb, called EWSR1, and that those on chromosome 11 are spread over a larger region of 40-50 kb, called EWSR2. Cosmids from loci flanking or overlapping these two regions have been obtained. We demonstrate here that fluorescence in situ hybridization (FISH) with these cosmids allows the localization of the two breakpoints with a 10-kb resolution and leads to a rapid and reliable ES/PNE diagnosis.

Precise localization on chromosome 12 of the ATF-1 gene by fluorescence in situ hybridization.

The ATF-1 gene encodes for a transcription factor normally regulated by cAMP (Hai et al. 1989, Yoshimura et al. 1990). Recently, it has been shown to be involved in the recurrent t(12;22) translocation observed in soft tissue malignant melanoma, in a fusion gene with the EWS gene (Zucman et al. 1993). We report here on its precise localization on chromosome 12 by fluorescence in situ hybridization.

Combinatorial generation of variable fusion proteins in the Ewing family of tumours.

Balanced translocations involving band q12 of human chromosome 22 are the most frequent recurrent translocations observed in human solid tumours. It has been shown recently that this region encodes EWS, a protein with an RNA binding homologous domain. In Ewing's sarcoma and malignant melanoma of soft parts, translocations of band 22q12 to chromosome 11 and 12 result in the fusion of EWS with the transcription factors FLI-1 and ATF1, respectively. The present analysis of 89 Ewing's sarcomas and related tumours show that in addition to the expected EWS-FLI-1 fusion, the EWS gene can be fused to ERG, a transcription factor closely related to FLI-1 but located on chromosome 21. The position of the chromosome translocation breakpoints are shown to be restricted to introns 7-10 of the EWS gene and widely dispersed within introns 3-9 of the Ets-related genes. This heterogeneity generates a variety of chimeric proteins that can be detected by immuno-precipitation. On rare occasions, they may be associated with a truncated EWS protein arising from alternate splicing. All 13 different fusion proteins that were evidenced contained the N-terminal domain of EWS and the Ets domain of FLI-1 or ERG suggesting that oncogenic conversion is achieved by the linking of the two domains with no marked constraint on the connecting peptide.

Regional fine mapping of the beta crystallin genes on chromosome 22 excludes these genes as physically linked markers for neurofibromatosis type 2.

Neurofibromatosis type 2 (NF2) is a rare autosomal dominant disease, characterized by the development of bilateral vestibular schwannomas. The NF2 gene has been assigned to chromosome 22. Cataract and other eye abnormalities are frequently seen in NF2 patients. The specific association of eye abnormalities and NF2 might be caused by a genetic change on chromosome 22 that affects both the NF2 gene and a physically linked crystallin gene. In order to test this hypothesis, we regionally localized the known crystallin genes (i.e. CRYBB2, CRYBB2P1, CRYBB3, and CRYBA4) on chromosome 22. Crystallin gene-specific probes were hybridized to an extended panel of human x rodent somatic cell hybrids containing various portions of chromosome 22. It was found that all crystallin genes map to a very small region on chromosome 22 that is physically separate from the NF2 gene region by at least 160 kb of DNA. In addition, we found that the beta B crystallin genes (CRYBB2, CRYBB2P1, and CRYBB3) are clustered on a 300 kb SacII fragment and that the beta A4 crystallin gene (CRYBA4) is not part of this cluster. We conclude that the ocular manifestations in many NF2 patients are probably not the primary consequence of rearrangements on chromosome 22 that involve both the NF2 gene and a nearby beta crystallin gene.