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Biomedical subjects

H Kovar

Publications and source records attributed to H Kovar.

At least 19 recordsLinked to original sources
PubMed2006

Suppression of KCMF1 by constitutive high CD99 expression is involved in the migratory ability of Ewing's sarcoma cells.

High CD99 expression levels and rearrangements of the EWS gene with ETS transcription factor genes characterize the Ewing's sarcoma family of tumors (ESFT). CD99 is a cell surface glycoprotein whose engagement has been implicated in cell proliferation as well as upregulation and transport of several transmembrane proteins in hematopoietic cells. In ESFT, antibody ligation of CD99 induces fast homotypic cell aggregation and cell death although its functional role in these processes remains largely unknown. Here, using an RNAi approach, we studied for the first time the consequences of modulated CD99 expression in six different ESFT cell lines, representing the most frequent variant forms of EWS gene rearrangement. CD99 suppression resulted in growth inhibition and reduced migration of ESFT cells. Among genes whose expression changes in response to CD99 modulation, the potassium-channel modulatory factor KCMF1 was consistently upregulated. In a series of 22 primary ESFT, KCMF1 expression levels inversely correlated with CD99 abundancy. Cells forced to express ectopic KCMF1 showed a similar reduction in migratory ability as CD99 silenced ESFT cells. Our results suggest that in ESFT, high CD99 expression levels contribute to the malignant properties of ESFT by promoting growth and migration of tumor cells and identify KCMF1 as a potential metastasis suppressor gene downregulated by high constitutive CD99 expression in ESFT.

12E7 Antigen↗
PubMed2004

Intraoperative dissemination of tumour cells in patients with Ewing tumours detected by RT-PCR.

Using reverse transcriptase polymerase chain reaction (RT-PCR) we evaluated the occurrence of tumour-cell ribonucleic acid (RNA) in the blood during surgery in patients with Ewing tumours. The patients received irradiation and chemotherapy according to the protocol of the European Intergroup Cooperative Ewing Sarcoma Study (EICESS) 92. Blood samples were taken from 15 patients. Intra-operative dissemination was found during 2/8 resections but showed no relation to patient survival. At second-look biopsy, detection of tumour-cell RNA was associated with relapse and metastases in 3/4 patients. The results suggest that pre-operative treatment did not completely prevent dissemination of tumour cells during surgery of Ewing tumours.

Adolescent↗
PubMed2003

A small nuclear RNA, hdm365, is the major processing product of the human mdm2 gene.

mdm2 encodes for an E3 ubiquitin ligase targeting constitutively expressed p53 for proteasomal degradation. Several protein isoforms have been described for human MDM2 (HDM2), some of which may correspond to splicing variants detectable by RT-PCR in many tumors. Upon cellular stress, p53 becomes resistant to MDM2 and, in a feedback loop, up-regulates mdm2 transcription. The physiological relevance of stress-induced mdm2 gene activity is not well understood. We describe a small nuclear RNA of 365 bases comprised of the first five hdm2 exons and lacking polyadenylation. hdm365 precedes full-length hdm2 RNA expression after induction by p53 and accumulates to significant levels in the nucleus, detectable at the site of hdm2 transcription and processing only. Considering a 10-fold lower stability and high steady-state levels of the novel RNA species, hdm365 appears to be the major processing product of hdm2 transcripts. hdm365 induction was observed after ectopic expression of p53 and after DNA damaging treatment of tumor cell lines, primary fibroblasts and lymphocytes, and was not related to apoptosis. Corresponding truncated transcripts were observed in hdm2 amplified cells. High stress-inducible expression levels, absence of a corresponding protein, and nuclear localisation of hdm365 suggest a novel RNA-based function for hdm2.

3T3 Cells↗
PubMed2001

The EWS protein is dispensable for Ewing tumor growth.

EWS encodes a ubiquitously expressed RNA binding protein with largely unknown function. In Ewing sarcoma family tumors (EFT), one allele is rearranged with an ETS gene. This is the first description of an EFT with a complete EWS deficiency in the presence of two copies of a rearranged chromosome 22 carrying an interstitial EWS-FLI1 translocation. Absence of EWS protein suggested that it is dispensable for EFT growth. By sequencing of EWS cDNA from unrelated EFTs, we excluded inactivation of EWS as a general mechanism in EFT pathogenesis. Rather, EWS was found to be uniformly expressed in two splicing variants of similar abundancy, EWSalpha and EWSbeta, which differ in a single amino acid. Three EWS negative cell lines were established, which will serve as valuable models to study normal and aberrant EWS function upon reintroduction into the tumor cells.

Alleles↗
PubMed2001

Intratumoural heterogeneity of 1p deletions and MYCN amplification in neuroblastomas.

BACKGROUND: At least three genetic hallmarks identify aggressive tumour behaviour in neuroblastomas; amplification of the oncogene MYCN; deletion (loss of heterozygosity [LOH]) at the short arm of chromosome 1 (del1p36), seen in approximately 28% of the cases; and di-tetraploidy. The MYCN oncogene is amplified in approximately 23% of all neuroblastomas and becomes important for the stratification of therapy in localised and 4s tumours. Up to now, it has been believed that the genetic constellation of neuroblastic tumours is stable and does not alter during tumour evolution or during tumour progression. PROCEDURE: Using fluorescence in situ hybridisation techniques (FISH) to investigate different tumour areas on touch preparations and histological sections, we show that genetic heterogeneity can be detected in neuroblastomas, especially in tumours detected by urinary mass screening. CONCLUSION: The identification of such cell clones is important, because the MYCN amplification and/or the deletion at 1p36 appear to be responsible for aggressive local growth and development of metastases.

Chromosomes, Human, Pair 1↗
PubMed2001

Two-dimensional DNA electrophoresis identifies novel CpG islands frequently coamplified with MYCN in neuroblastoma.

BACKGROUND: Amplification of the oncogene MYCN in neuroblastoma has been found to correlate with aggressive tumour growth and is used as a predictor of clinical outcome. The MYCN amplicon is known to involve coamplification of extensive DNA regions. Therefore it is possible that other genes are coamplified in this amplicon and that they may play a role in the poor outcome of MYCN amplified tumours. PROCEDURE: We have implemented an approach for the two-dimensional separation of human genomic restriction fragments to detect and isolate as yet unknown amplified sequences in the MYCN amplicon in neuroblastoma. Using this approach we have recently cloned a novel gene referred to as NAG that is frequently coamplified with MYCN in neuroblastoma. RESULTS AND CONCLUSIONS: We report here the identification and cloning of two additional CpG islands that are amplified in neuroblastoma. One contains a sequence that is identical to the first intron of DDX1. The other represents a novel CpG island that is associated with an as yet unidentified gene. We show that the novel CpG island is located in close proximity to the MYCN locus on chromosome 2 and is as frequently coamplified with MYCN in neuroblastoma as NAG and DDX1.

Chromosomes, Human, Pair 1↗
PubMed2001

Fine-mapping of cytogenetically undetectable EWS/ERG fusions on DNA fibers of Ewing tumors.

In contrast to the EWS/FLI1 fusion which is represented by a t(11;22)(q24;q12), EWS/ERG fusions are frequently cytogenetically not detectable. Three Ewing tumors (ET), two with apparently normal chromosomes 21 and 22, and one ET with a t(2;22)(p25;q12), were studied by FISH on interphase nuclei, metaphase chromosomes and on DNA fibers. EWS/ERG transcripts were detected by RT-PCR in all cases. FISH, using cosmids located proximally (F10, G9) and distally (F7) to the EWS breakpoint region, revealed no detectable separation of these probes in two cases. In contrast, co-hybridization of probe PT1526 containing the ERG breakpoint region with G9 revealed the juxtaposition of two signals per interphase nucleus in all three cases indicating the EWS/ERG fusions. Chromosome preparations displayed the juxtaposed signals on the der(22), and hybridization signals of the probes PT1526 and G9 on the non-rearranged chromosomes 21 and 22 in all cases, respectively. The PT1526 signal on the der(21) was seen only in cases 1 and 2. These results were confirmed by triple-target FISH on tumor DNA fibers. In all three cases, the hybridization pattern F10 - G9 - PT1526 indicates a centromere to telomere orientation. This finding suggests that EWS/ERG fusions in ETs may be generated by an inversion of the ERG gene or a part thereof followed by an insertion into the EWS gene on the der(22). Double-target FISH on interphase nuclei using probes flanking the EWS breakpoint region and probe PT1526 enables the detection of virtually all 22q12 rearrangements in ETs, thus providing a reliable diagnostic assay.

Cation Transport Proteins↗
PubMed2000

Characterization of distinct consecutive phases in non-genotoxic p53-induced apoptosis of Ewing tumor cells and the rate-limiting role of caspase 8.

To dissect the p53-dependent apoptotic pathway, events following induction of temperature sensitive (ts) p53val138 were studied in a Ewing tumor cell line. Transcriptional deregulation of p53 targets first observable after 1 h at 32 degrees C preceded activation of caspases and the break-down of mitochondrial respiratory activity. Activation of caspases was first observed 4 h after p53 induction. Using peptide inhibitors we identified activation of caspase 8 upstream of caspases-9 and -3. Although the caspase 8 specific inhibitor z-IETD.fmk did not affect translocation of BAX to the mitochondrial membrane and cytochrome C release it almost completely blocked cleavage of the prototype caspase substrate PARP and DNA fragmentation while enforcing mitochondrial depolarization and production of reactive oxygene species (ROS). Activation of caspase 8 did not involve death-domain receptor signaling. Expression of BCL2 only partially suppressed caspase activation but blocked apoptosis. Replacement of the N-terminus of p53val138 by the related VP16 transactivation domain created a ts p53 with a tanscriptional activity indistinguishable from p53val138 until the time of caspase activation. However, the VP16 - p53 fusion failed to trigger caspases and subsequent induction of the ROS producing gene pig3 paralleled by complete loss of apoptotic activity. These results indicate that p53-dependent transcriptional deregulation, triggering of the caspase cascade and the mitochondrial break-down occur in a timely ordered sequence coordinated by the genuine p53 amino terminus and suggest caspase 8 and PIG3 as key regulatory elements in this process. Oncogene (2000) 19, 4096 - 4107

Apoptosis↗
PubMed2000

Adenovirus E1A does not induce the Ewing tumor-associated gene fusion EWS-FLI1.

Rearrangement of the EWS gene with FLI1 is thought to occur early in the pathogenesis of Ewing's sarcoma family tumors (EFTs) because the chromosomal aberration is pathognomonic for this disease. Recently, adenovirus (Ad) 5 E1A protein has been reported to induce this gene rearrangement in a variety of cell types. This finding, if generally substantiated, not only suggests an etiological role for viral agents in the generation of oncogenic chromosomal aberrations but would also significantly impact the use of adenoviral vectors for gene therapy. In contrast, we now report on the absence of EWS-FLI1 chimeric products from short- and long-term cultures of stably Ad-transformed cells lines and from transiently E1A-expressing cell lines. In addition, we demonstrate the absence of E1A from EFTs. We conclude that there is no role for Ads in EFT pathogenesis. Consequently, evidence for a viral genesis of tumor-specific gene rearrangements is not available.

Adenovirus E1A Proteins↗
PubMed2000

Variability in gene expression patterns of Ewing tumor cell lines differing in EWS-FLI1 fusion type.

Type 1 and type 2 EWS-FLI1 fusion products result from variation in breakpoint locations arising from the t(11;22)(q24;q12) recurrent chromosomal translocation in Ewing's sarcoma family tumors (EFT). Previously, studies from our institution (updated in the present communication at a median follow-up of more than 6 years) and others suggested a prognostic difference for EFT patients with localized disease depending on the type of EWS-FLI1 fusion present in the tumor. It has been suggested that the observed clinical discrepancies result from different transactivation potentials of the various EWS-FLI1 fusion proteins. In an attempt to identify genes whose expression levels are differentially modulated by structurally different EWS-FLI1 transcription factors, we have used two related PCR-based subtractive approaches, cDNA representational difference analysis (cDNA-RDA) and linker-capture subtraction (LCS) to compare transcript representations in cDNA pools of type 1 versus type 2 EFT cell lines. About 800 clones obtained by the two approaches were analyzed by dot blot hybridization to cDNA pools. Eighty-six clones showing the highest variability in signal intensities on the dot blots were further hybridized to individual EFT cell line RNAs on Northern blots, and four of them were additionally studied by real-time quantitative PCR (RTQ-PCR). Although interindividual variations in gene expression patterns in the range of one- to several-fold were observed, no correlation to specific EWS-FLI1 fusion types could be identified. Among the genes differentially expressed in individual EFT cell lines are several previously implicated in tumor growth, invasion, and metastasis. Although our data may have revealed candidate genes whose composite expression pattern may be relevant for the biology of individual EFT, they do not support a role of distinct EWS-FLI1 fusion types for EFT prognosis based on different transactivation potentials.

Blotting, Northern↗
PubMed1999

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.

Adolescent↗
PubMed1999

Co-amplification of a novel gene, NAG, with the N-myc gene in neuroblastoma.

Substantial evidence implicates amplification of the N-myc gene with aggressive tumor growth and poor outcome in neuroblastoma. However some evidence suggests that this gene alone is not the sole determinant of outcome in N-myc amplified tumors. We have searched for genes that co-amplify with N-myc in neuroblastoma by means of two-dimensional analysis of genomic restriction digests. Using this approach, we have identified and cloned a novel genomic fragment which is co-amplified with N-myc in neuroblastomas. This fragment was mapped in close vicinity to N-myc on chromosome arm 2p24. It was amplified in 5/8 N-myc amplified neuroblastoma cell lines and in 9/13 N-myc amplified tumors. Using a PCR-based approach we isolated a 4.5 kb c-DNA sequence that is partly contained in the genomic fragment. The open reading frame of the cDNA encodes a predicted protein of 1353 amino acids (aa). The homology of the predicted protein, which we designated NAG (neuroblastoma amplified gene), to a C. elegans protein of as yet unknown function, and its ubiquitous expression suggest that NAG may serve an essential function. By Northern blot analysis we showed that amplification of the cloned gene correlates with over-expression in neuroblastoma cell lines. Amplification and consequent over-expression of NAG may, therefore, contribute to the phenotype of a subset of neuroblastomas.

Chromosome Mapping↗
PubMed1999

Relation of neurological marker expression and EWS gene fusion types in MIC2/CD99-positive tumors of the Ewing family.

The Ewing family of tumors (EFT) is characterized by high MIC2/CD99 expression and specific EWS/ETS gene rearrangements, resulting in different chimeric transcripts. Further division into peripheral primitive neuroectodermal tumors and Ewing's sarcoma is still debated and, in the absence of distinct morphological parameters, has been based on the reactivity with neuroglial markers (NgM). We investigated 44 EFT in terms of a possible correlation between the type of EWS chimeric transcripts and reactivity with the following NgM: polyclonal and monoclonal neuron-specific enolase (NSE), S-100, chromogranin A, synaptophysin, Leu-7, glial fibrillary acid protein, and neurofilament. EWS/Fli1 fusion type 1 was detected in 30 of 44 and type 2 in 11 of 44 tumors. Three tumors, presenting with an uncommon morphology, carried rare chimeric transcripts. Our results indicate an association of lack of NgM staining with type 1 EWS/Fli1 translocations, found in 16 of 18 tumors with no NgM expression as detectable by any of the antibodies we applied. Using the monoclonal NSE antibody, 21 of 26 tumors without NgM staining expressed type 1 EWS/FLI1chimeric RNA, whereas in the groups with 1 or more and 2 or more NgM, only 9 of 17 and 1 of 5 tumors, respectively, carried type 1 EWS/Fli1 fusion transcripts. Despite this association of increased NgM expression with a non-type 1 EWS/Fli1 gene fusion, a strict correlation between the extent of NgM expression and certain EWS fusion types was not evident. This fortifies the concept to consider EFT as a spectrum of tumors and suggests the type of EWS fusion transcripts as one, but not the only parameter influencing the extent of differentiation.

Adolescent↗
PubMed1999

Low incidence of molecular evidence for tumour in PBPC harvests from patients with high risk Ewing tumours.

Reverse transcriptase polymerase chain reaction (RT-PCR) was applied to evaluate the frequency of tumour cells in PBPC products from 15 high risk Ewing tumour (ET) patients who were treated according to EICESS 92 with high-dose chemotherapy (HDC) and stem cell rescue. Initial tumour cell contamination of the bone marrow (BM) detected by light microscopy was found in five and by RT-PCR in eight cases. RT-PCR was performed on each PBPC sample repeatedly at a sensitivity comparable to 20-100 highly EWS-Fli1 expressing tumour cells per 10 ml of fresh blood. Irrespective of the extent of BM involvement at diagnosis, all BM samples obtained before harvest were RT-PCR negative. Among 12 of 35 analysed apheresis products with single positive RT-PCR results only one sample tested reproducibly positive for tumour cell contamination in independent determinations. These preliminary data suggest that tumour cell contamination of PBPC is rarely found in patients with ET.

Adolescent↗
PubMed1999

The Ewing family of tumors and the search for the Achilles' heel.

The ideal cancer therapy would accomodate the specific biology of a tumor and be based upon understanding the mechanisms of malignancy. This vision has been the driving force in cancer research. However, the story of success in clinical cancer management is a story of empirie largely independent from progress in basic research. For the Ewing family of tumors (EFT) comprising Ewing's sarcoma and peripheral primitive neuroectodermal tumor, significant insights into the molecular basis have appeared recently. Some of last year's discoveries may have taken us closer to the identification of the Achilles' heel in this disease. The first clue has been obtained to the mechanism of chromosomal translocation, which constitutes a rate-limiting step in EFT pathogenesis. Also, researchers have progressed in understanding the control of EFT cell proliferation, differentiation, and death. A major role in these processes has been attributed to the EWS-ets gene rearrangement. Specific growth factor circuits appear to be involved in deregulated tumor cell growth. By analogy to heterologous cellular systems, it is possible to postulate an important functional role for CD99(MIC2) as it contributes to the malignant phenotype of EFT. In vitro, as well as the first in vivo, experimental evidence suggests that tumor cell expansion and spread can be counteracted by breaking these physiological pathways. Still, we are far from a tailored biological therapy of EFT. Before this goal may be achieved, we must seek further improvements and diversification of today's standard and intensified treatment regimens.

Animals↗
PubMed1999

CD99 positivity and EWS-FLI1 gene rearrangement identify a breast tumor in a 60-year-old patient with attributes of the Ewing family of neoplasms.

Rearrangements of the EWS gene with ETS transcription factor genes as a result of chromosomal translocation and high expression levels of CD99MIC2 characterize the Ewing family of tumors (EFT). This group of rather undifferentiated neoplasms affects bone and soft tissue in children and young adults mostly between 5 and 30 years of age (median, 15 years). This study reports a case of a CD99MIC2 positive small round cell tumor in the breast of a 60-year-old woman in whom a t(11;22)(q24;q12) chromosomal aberration was identified by cytogenetic analysis. Reverse transcriptase (RT)-polymerase chain reaction (PCR) followed by sequence analysis revealed expression of a chimera transcript in which EWS exon 10 was fused to FLI1 exon 6. Previously, this gene fusion has been reported to occur in approximately 3% of EFT. The specific gene rearrangement of EWS intron 10 was confirmed on Southern blot of genomic DNA. This study further contributes to the growing list of unusual neoplasms in adults that carry genotypic and phenotypic traits of the EFT.

12E7 Antigen↗
PubMed1999

EWS-FLI1 and EWS-ERG gene fusions are associated with similar clinical phenotypes in Ewing's sarcoma.

PURPOSE: There are a variety of solid tumors in which alternative chromosomal translocations generate related fusion products. In alveolar rhabdomyosarcoma and synovial sarcoma, these variant fusions have been found to have major clinical significance. We investigated whether the two alternative gene fusion products, EWS-FLI1 and EWS-ERG, define different clinical subsets within the Ewing's sarcoma family of tumors. PATIENTS AND METHODS: We selected 30 cases of Ewing's sarcoma with the EWS-ERG gene fusion and 106 cases with the EWS-FLI1 fusion. Clinical data were obtained for each case and compared with the molecular diagnostic findings. RESULTS: There were no significant clinical differences observed between the two groups in age of diagnosis, sex, metastasis at diagnosis, primary site, event-free survival, or overall survival. CONCLUSION: Differences in the C-terminal partner in the Ewing's sarcoma family gene fusions are not associated with significant phenotypic differences.

Adolescent↗