Biomedical subjects

H Kovar

Publications and source records attributed to H Kovar.

At least 37 records · Page 2Linked to original sources

PubMed1998

Oncogenic EWS-Fli1 interacts with hsRPB7, a subunit of human RNA polymerase II.

As a result of the t(11;22)(q24;q12) chromosomal translocation characterizing the Ewing family of tumors (ET), the amino terminal portion of EWS, an RNA binding protein of unknown function, is fused to the DNA-binding domain of the ets transcription factor Fli1. The hybrid EWS-Fli1 protein acts as a strong transcriptional activator and, in contrast to wildtype Fli1, is a potent transforming agent. Similar rearrangements involving EWS or the highly homologous TLS with various transcription factors have been found in several types of human tumors. Employing yeast two-hybrid cloning we isolated the seventh largest subunit of human RNA polymerase II (hsRPB7) as a protein that specifically interacts with the amino terminus of EWS. This association was confirmed by in vitro immunocoprecipitation. In nuclear extracts, hsRPB7 was found to copurify with EWS-Fli1 but not with Fli1. Overexpression of recombinant hsRPB7 specifically increased gene activation by EWS-chimeric transcription factors. Replacement of the EWS portion by hsRPB7 in the oncogenic fusion protein restored the transactivating potential of the chimera. Our results suggest that the interaction of the amino terminus of EWS with hsRPB7 contributes to the transactivation function of EWS-Fli1 and, since hsRPB7 has characteristics of a regulatory subunit of RNA polymerase II, may influence promoter selectivity.

3T3 Cells↗

PubMed1998

The Ewing tumor family of peripheral primitive neuroectodermal tumors expresses human gastrin-releasing peptide.

The Ewing tumor family of peripheral primitive neuroectodermal tumors (pPNETs) are characterized by chromosomal translocations leading to EWS-ETS gene fusions. These hybrid genes express chimeric proteins that are thought to act as aberrant transcription factors. We therefore used differential display-PCR to compare gene expression patterns in pPNET cell lines with those of other small round cell tumors (SRCTs) of childhood. This technique detected differential expression of sequences corresponding to human gastrin-releasing peptide (GRP) in pPNET cell lines but not in other SRCT cell lines. Subsequent Northern and reverse transcription-PCR analysis of SRCT cell lines confirmed GRP positivity in all pPNET lines tested. Of primary tumors tested by reverse transcription-PCR, GRP expression was found in 7 (44%) of 16 pPNETs but in no other primary SRCTs examined. Expression of the GRP receptor gene was demonstrable in 55% of pPNET cell lines and 25% of primary pPNET tumors but also in several other SRCTs. Radioimmunoassays and immunohistochemistry confirmed expression of bioactive GRP peptide in pPNET cell lines and primary tumors, respectively. Moreover, in vitro growth of a pPNET cell line was slowed by treatment with a GRP receptor antagonist and accelerated by a GRP receptor agonist. GRP is a known autocrine growth factor in small cell lung cancer and other neuroendocrine tumors. Its expression in pPNETs provides further evidence for a neuroectodermal histogenesis of these tumors and suggests that autocrine growth of this family of tumors may be at least partially regulated by GRP.

Artificial Gene Fusion↗

PubMed1998

A recombination based method to rapidly assess specificity of two-hybrid clones in yeast.

The yeast two-hybrid system is frequently used to identify protein-protein interactions. Confirming the specificity of candidate clones requires separation and isolation of yeast plasmids, propagation in bacteria and testing combinations of DNA-binding and activation domain hybrids in yeast. In order to simplify this procedure, we developed a rapid method based on PCR amplification of library insert DNAs and in vivo cloning into the activation domain hybrid vector. Reporter gene activity is assayed in parallel for combinations with different DNA-binding domain hybrids. Further characterization of inserts does not require plasmid isolation and intermediate hosts.

Antigens, Viral, Tumor↗

PubMed1998

Cloning of a novel human ELF-1-related ETS transcription factor, ELFR, its characterization and chromosomal assignment relative to ELF-1.

The ETS gene family encodes a group of proteins that function as transcription factors under physiological conditions and, if aberrantly expressed, can lead to cellular transformation. ETS transcription factors are characterized by a unique conserved DNA binding domain. A subset of these proteins is rearranged with EWS in Ewing tumors (ET). We recently described a spectrum of ETS genes coexpressed with EWS-FLI1 in an ETcell line to define proteins that potentially compete in target site selection. We now report on the cloning and characterization of a novel ETS family member, ELFR, displaying 92% homology to ELF-1 in its DNA binding domain while diverging in the rest of the protein. ELFR expression was found in a very tissue restricted pattern with the highest abundancy in placenta. We also report the chromosomal assignment of ELFR and ELF-1 to Xq26 and 13q13, respectively, by means of fluorescence in-situ hybridization (FISH).

Amino Acid Sequence↗

PubMed1998

Predictive potential of testing for bone marrow involvement in Ewing tumor patients by RT-PCR: a preliminary evaluation.

EWS/ets-oncogene fusion transcripts can be detected in at least 98% of Ewing tumors [(ET) Ewing sarcoma and peripheral primitive neuroectodermal tumor] by reverse transcriptase-polymerase chain reaction (RT-PCR), thus confirming the histopathologic diagnosis. To detect minimal amounts of tumor cells in the bone marrow (BM), we used an RT-PCR assay with a high sensitivity, revealing one tumor cell in a background of 10(6) normal cells. We examined BM samples from 35 newly diagnosed ET patients (23 with localized and 12 with metastatic disease). At diagnosis, tumor cells in the BM were detected in 7/23 patients with localized disease (30%). Fifty percent of patients with isolated lung metastasis were RT-PCR positive (3/6), whereas 6/6 patients with bone metastases showed positive signals (100%). All patients with initial PCR positivity in the BM became negative during treatment. After a median follow-up of 30 months, relapses were observed in both groups of patients with localized disease (3/7 RT-PCR positive and 2/16 RT-PCR negative). The only recurrence in the group with isolated lung metastases occurred as progressive lung disease in 1 of the 2 RT-PCR-negative patients, whereas among the 6 patients with bone metastases 2 remain in complete remission. So far, RT-PCR screening for BM involvement did not allow prediction of early relapse in ET. To assess better the significance of this test in the evaluation of long-term prognosis and in monitoring the effectiveness of systemic therapy, long observation periods are warranted before it becomes a tool for treatment stratification.

Adolescent↗

PubMed1998

Use of transcription reporters with novel p53 binding sites to target tumour cells expressing endogenous or virally transduced p53 mutants with altered sequence-specificity.

p53 triple mutants (120N/121G/277H, 120H/121G/ 277H, 120S/121G/277H and 120H/121G/277Y) have altered sequence specificity in bandshift assays in vitro and transcription assays in vivo. These mutants activate transcription from the site TTT CATG AAA but not from wild type sites. The triple mutants activate more strongly than p53 with a single 277Y mutation. The TTT site matches the wild type p53 consensus at only 4/10 positions and is not recognised by wild type p53. 277Y mutations have been described in human tumours, and Ewing tumour cells expressing this mutant from the endogenous p53 locus selectively activate transcription from transfected luciferase reporters regulated by TTT-mutant p53 binding sites. p53 mutants with altered sequence specificity have potential advantages for cancer gene therapy: if used to activate transcription of conditionally toxic genes they would allow tumour-targeting by p53, which acts as a sensor for the malignant state, but place control over cell killing in the hands of the clinician. Rare tumours expressing such mutants from the endogenous p53 locus could be targeted directly with p53-regulated suicide vectors, but for most tumours both the p53 mutant and the reporter would need to be encoded by the virus.

Base Sequence↗

PubMed1998

[Cytogenetic and molecular genetic changes in malignant primary bone tumors].

Osteosarcoma, chondrosarcoma and tumors of the Ewing group are the most frequently observed primary malignant bone tumors. In an Internet homepage recently constructed for the Orthopedic Hospital Rizzoli Bologna, Italy, these tumors have represented the majority of 4423 malignant bone tumors in the archives of this institution since 1920 (http:/(/)www.tizeta.it/rizzoli). Malignant fibrous histiocytoma, fibrosarcoma, hemangioendothelioma, malignant hemangiopericytoma and giant-cell tumors are diagnosed less frequently. Since the introduction of modern molecular and cytogenetic techniques, knowledge of genetic aberrations in malignant bone tumors has steadily increased. However, so far only for the group of Ewing tumors has a recurrent chromosomal marker, the translocation t(11;22) (q24;q12), been identified.

Bone Neoplasms↗

PubMed1998

Ewing's sarcoma and peripheral primitive neuroectodermal tumors after their genetic union.

The Ewing family of tumors has been defined by the presence of an EWS-ets gene rearrangement. Using molecular techniques for the visualization of this aberration, several tumors of unusual location and histology were recently identified and added to this group. Increasing experimental data suggest that EWS-ets fusions act as transforming transcription factors. Subtractive screening of transgenic fibroblasts has led to the identification of potential targets. The authentic cellular context for these aberrant gene products is still unknown and may be diverse, however. Clinical heterogeneity has stimulated a search for genetic factors that may relate to treatment response. The effect of the EWS-ets fusion transcript structure on localized disease has been recently confirmed and frequent p16 tumor suppressor alterations have been described. The presence of metastases at diagnosis as the major predictor of outcome has been assessed on a submicroscopic level by reverse transcriptase-polymerase chain reaction. Problems arising from these studies are discussed, although the biological basis for variable disease extension remains obscure.

Heterogeneous-Nuclear Ribonucleoproteins↗

PubMed1997

Neuroblastoma screening in infants postponed after the sixth month of age: a trial to reduce "overdiagnosis" and to detect cases with "unfavorable" biologic features.

BACKGROUND: Encouraged by Japanese reports of the benefits of screening 6 month-old infants for neuroblastoma, a neuroblastoma screening program was introduced in Austria in 1991. However, because of concerns related to "overdiagnosis" by screening at this age, the screening test was performed at a later age. METHODS: From March 1991 to February 1995 neuroblastoma screening was performed on filter paper urine specimens in 100,043 Austrian infants (median age 8.5 months). Primary analysis of urine catecholamines (vanillylmandelic acid and homovanillic acid was performed by use of an E1A method. Questionable or positive results were confirmed by high performance liquid chromatography (HPLC). A double retest was requested following a positive HPLC result. RESULTS: Twenty-one infants were admitted to a hospital following repeatedly elevated values of vanillymandelic acid (VMA) and/or homovanillic acid (HVA). Eleven infants were found to have neuroblastoma (three stage 1, four stage 2B, four stage 3). Treatment consisted of surgery alone with total or subtotal resection in eight cases, surgery and chemotherapy in two cases, and chemotherapy alone in one case. Biologic features were assessed in all tumors excluding ploidy in one case. The majority of the tumors analyzed were near-triploid (9/10), however, two tumors revealed N-myc amplification. CONCLUSIONS: Our results demonstrate that stage distribution and biologic features of neuroblastomas diagnosed by screening at 8.5 months are different from the results of screening at 6 months. Furthermore, the detection of one neuroblastoma among 9,100 screened infants is significantly lower than the incidence of the Japanese screening program. Our results suggest that screening at an age of 7 to 10 months reduces overdiagnosis and may be of more benefit than earlier screening.

Age Factors↗

PubMed1997

Acute leukemias express a functional receptor for the human growth hormone.

The potential influence of the human growth hormone (hGH) on the behavior of acute leukemias is a matter of controversy. We investigated primary childhood and adult leukemias (n = 44) and leukemic cell lines (n = 13) for the expression of the hGH receptor (hGHR) by immunohistochemistry and flow cytometry. All leukemias expressed the hGHR in the cytoplasm; expression on the surface was undetectable in some of the leukemias. In leukemic cell lines, hGHR expression on the surface was demonstrated in a dose-dependent manner after incubation with rhGH. Physiologic concentrations of hGH were more efficient than higher doses in increasing hGHR surface expression. A proliferative response to hGH was accomplished in cell lines REH, Molt4, and K562. However, only one of 19 primary leukemias (ALL, n = 12; AML, n = 7) showed increased cell counts after the addition of 50-800 ng/ml recombinant hGH (rhGH). These cells were of an immature T-cell phenotype. We thus conclude that acute leukemias can be stimulated by hGH to up-regulate its receptor, but that most primary leukemias may require additional signals for the induction of proliferation.

Adult↗

PubMed1997

Neuroblastoma cells can actively eliminate supernumerary MYCN gene copies by micronucleus formation--sign of tumour cell revertance?

Human neuroblastoma cell lines frequently exhibit MYCN amplification and many are characterised by the presence of morphologically distinct cell types. The neuronal cells (N-cells) and the so-called flat cells (F-cells) are thought to represent manifestations of different neural crest cell lineages and are considered to be the consequence of neuroblastoma cell pluripotency. In this study, various neuroblastoma cell lines were examined for micronuclei. In F-cells of neuroblastoma cell lines with extrachromosomally amplified MYCN, we observed the frequent occurrence of micronuclei. Using fluorescence in situ hybridisation (FISH) with a MYCN specific probe, we demonstrated that these micronuclei were packed with MYCN hybridisation signals. In addition, in a minor percentage of cells, MYCN signals occurred in clusters, adhered to the nuclear membrane and aggregated in nuclear protrusions. In F-cells, a substantial reduction or lack of amplified MYCN copies was observed. These observations let us conclude that extrachromosomally amplified genes can be actively eliminated from the nucleus resulting in a dramatic loss of amplified sequences in the F-cells. Moreover, reduction or loss of amplified sequences in F-cells was shown to be accompanied by downregulation of MYCN expression, by a decrease in proliferative activity and by upregulation of molecules of the major histocompatibility complex class I (MHC I). Interestingly, F-cells are not restricted to neuroblastoma cell cultures, but also occur in cell lines of other tissue origin. All F-cells share important biological features, interpreted as cell revertance, i.e. loss of the malignant phenotype and properties. This fact, together with the demonstration that neuroblastoma cells do not differentiate into Schwann cells in vivo [1] Ambros et al. NEJM 1996, 334, 1505-1511, do not support the hypothesis that F-cells represent Schwannian/glial differentiation in vitro. We therefore postulate that the elimination of amplified MYCN gene copies in cultivated neuroblastoma cells is in line with the phenomenon of tumour cell revertance.

Cell Division↗

PubMed1997

Among genes involved in the RB dependent cell cycle regulatory cascade, the p16 tumor suppressor gene is frequently lost in the Ewing family of tumors.

The pRB cell cycle regulatory cascade is frequently perturbed in neoplasia by overexpression of a component of the pRB-phosphorylating cyclin D1/CDK4 complex or by inactivation of pRB or the CDK4 inhibitors p16 and p15. We investigated the status and expression of p16, p15, CCND1, CDK4 and RB genes in the Ewing family of tumors. P16 loss was observed in 8 of 27 tumors (30%) and in 12 of 23 (52%) tumor cell lines from unrelated patients. There were no discrepancies in the p16 status between primary tumors and the corresponding cell lines and between cell lines established from consecutive tumor samples. p15 was codeleted in most cases but p15 mRNA was absent also in cell lines retaining the gene. In addition, posttranscriptional p16 inactivation was observed in two cases. Although no evidence for CDK4 or CCND1 amplification was obtained, expression of these genes varied considerably in the cell lines in a case specific manner. In wild-type p16 cell lines, pRB expression was lost in one case. Our data indicate that, despite the absence of cytogenetically detectable 9p21 chromosomal aberrations, p16 deletions constitute the most frequent secondary molecular aberration in Ewing tumors so far. These results are discussed in the context of the stage of disease and the clinical outcome of the patients. The potential prognostic impact of these findings remains to be further evaluated.

Cell Cycle↗

PubMed1996

Fas- and activation-induced apoptosis are reduced in human T cells preactivated in the presence of TGF-beta 1.

The elimination of activated but not resting T cells involves apoptosis induced either by restimulation via the TCR/CD3 complex, CD2, or by signaling through the Fas Ag. The factors regulating the shift of an apoptosis-resistant to an apoptosis-sensitive phenotype and vice versa have not so far been clarified. Here we report that TGF-beta 1, when present during a PHA activation course, significantly increases viability of human T cells upon reculture in medium alone, following restimulation via CD2, CD3, or after triggering the Fas Ag. Using DNA gel electrophoresis and an in situ nick translation technique we further show that activation-induced and Fas-mediated apoptosis are reduced in T cells that were prestimulated with PHA plus TGF-beta 1, compared with control cells prestimulated with PHA alone. Moreover, when PHA-preactivated T cells are further expanded in IL-2, inclusion of TGF-beta 1 results in higher cell yields at any timepoint from day 30 to 75 of cell culture compared with control cultures without TGF-beta 1. However, no differences in Fas or bcl-2 protein expression are found between cells stimulated in the absence or presence of TGF-beta 1. Together, our data identify TGF-beta 1, when present during an activation course, as an important viability factor possibly of importance for the generation of effector and/or long-lived memory T cells.

Apoptosis↗

PubMed1996

Mobilization of tumour cells during biopsy in an infant with Ewing sarcoma.

UNLABELLED: Ewing sarcoma and the closely related peripheral primitive neuroectodermal tumour, recently referred to as Ewing tumour (ET), are characterised by unique gene rearrangements on chromosome 22. The breakpoints have been cloned and shown to fuse the Ewing sarcoma gene to one of two closely related ETS proto-oncogens, FLI-1 or ERG, which reside on chromosomes 11 and 21, respectively. The rearrangement results in the expression of specific hybrid transcripts which can be detected with high sensitivity by the reverse transcriptase polymerase chain reaction technique (RT-PCR) in primary tumours, blood and bone marrow. We report on a 7-month-old boy with a pelvic Ewing sarcoma in whom circulating tumour cells were identified in the peripheral blood during open tumour biopsy by RT-PCR. However, before and 6 days after surgery no tumour cells could be detected in the peripheral blood. CONCLUSION: The application of RT-PCR to monitor shedding of tumour cells during surgical intervention will help to evaluate if open biopsy potentially contributes to metastatic tumour cell spread.

Base Sequence↗

PubMed1996

Metastatic extraosseous Ewing tumor. Association of the additional translocation der(16)t(1;16) with the variant EWS/ERG rearrangement in a case of cytogenetically inconspicuous chromosome 22.

In Ewing sarcoma and related tumors, recently referred to as the Ewing tumors (ET), t(11;22)(q24q12) and its molecular genetic equivalent, the EWS/FLI-1 rearrangement, characterize approximately 85% of cases, while variant aberrations are rare. A second nonrandom aberration in ET is the unbalanced t(1;16) accompanying the t(11;22) in roughly 17% of cases. We present a 17-year-old man with estraosseous ET and multiple metastases, in whom the only cytogenetically detectable chromosomal aberration was der (16)t(1;16)(q12;q11.2). This finding was confirmed by fluorescence in situ hybridization (FISH). Using the RT-PCR technique, a variant EWS/ERG fusion transcript was noted, resulting from a t(21;22) chromosomal rearrangement which recently demonstrated in roughly 10% of ET. However, data on possible biologic differences in EWS/FLI-1 versus EWS/ERG expressing ET are as yet unavailable. This is the first reported combination of t(1;16) with the EWS/ERG rearrangement. A possible significance of this finding for Ewing tumor progression is discussed.

Adolescent↗

PubMed1996

Variability of nm23-H1/NDPK-A expression in human lymphomas and its relation to tumour aggressiveness.

The nm23-H1 gene is a putative metastasis-suppressor gene encoding a 17 kDa protein with nucleoside diphosphate kinase activity. Expression of nm23-H1/NDPK-A correlates inversely with the metastasising potential of some human tumours and experimental animal cells. No nm23 expression studies exist for human malignant lymphomas so far. In this study, we examined nm23-H1 expression by Northern and immunohistochemical analysis in 106 primary lymphoma samples from patients with Hodgkin's disease (HD) (n = 15), high-grade non-Hodgkin's lymphoma (NHL) from different lineages (n = 71) and low-grade NHL (n = 20). Both inter- and intra-subtype variations in nm23-H1/NDPK-A expression levels were demonstrated by all disease subtypes. Besides this heterogeneity, a general trend towards highly malignant samples expressing higher nm23-H1/NDPK-A, levels than the low-grade lymphomas was observed. Both adult and childhood HD and high-grade NHL samples exhibited significantly higher NDPK-A expression than the low-grade NHL found only in adults. High nm23-H1/NDPK-A levels in lymphoma samples did not always reflect proliferative activity of tumour cells as monitored by Ki-67 antigen staining. Fifty samples were further investigated for possible mutations in the nm23-H1 coding sequence by means of reverse transcriptase-polymerase chain reaction (RT-PCR) and single-strand conformation polymorphism (SSCP) analysis. No mutation was found by this screening. Our results suggest a role for nm23-H1 expression in the disease aggressiveness of lymphomas.

Adolescent↗

PubMed1996

NPM/ALK gene fusion transcripts identify a distinct subgroup of null type Ki-1 positive anaplastic large cell lymphomas.

The chromosomal aberration t(2:5) resulting in the juxtaposition of NPM and ALK genes is a well-known feature of several Ki-1+ anaplastic large cell lymphomas (ALCL) of the T-cell type. However, conflicting results have been reported concerning the presence of this gene rearrangement in other ALCL and Hodgkin's disease (HD), respectively. We performed NPM/ALK RT-PCR on 14 cases of ALCL expressing distinct myelomonocytic markers, e.g. CD11c, CD13, CD14 or CD68, but neither T-cell nor B-cell associated antigens (null cell phenotype). The specific translocation was found exclusively in six childhood tumours previously diagnosed as malignant histiocytosis (MH), whereas all adult lymphomas (three ALCL without characteristics of MH, three secondary ALCL following HD) and two paediatric cases of secondary ALCL following HD did not show NPM/ALK gene fusion products. By Southern blotting, the status of T-cell receptor (TCR) and immunoglobulin heavy chain genes (IgH) were investigated; two patients with initially diagnosed MH had the TCRdelta-chain gene rearranged (Ddelta2-Ddelta3 and Vdelta1-Jdelta1, respectively). IgH rearrangements were detected in only one patient with secondary ALCL. Our data indicate a high association of previously diagnosed MH and NPM/ALK gene rearrangements. In one case, this specific translocation was demonstrated at an early stage of development; in another, a mature TCRdelta-chain gene rearrangement was detected. These data support the hypothesis of a lymphoid origin of this subgroup of Ki-1 positive ALCL previously diagnosed as MH.

Adolescent↗

PubMed1996

Does expression of different EWS chimeric transcripts define clinically distinct risk groups of Ewing tumor patients?

PURPOSE: Because of the high heterogeneity of EWS gene fusions with FLI1 and ERG genes due to variable chromosomal breakpoint locations in Ewing tumors (ET) (14 different chimeric transcripts identified so far), we evaluated the clinical impact of the expression of diverse fusion transcripts in ET patients. PATIENTS AND METHODS: In a European multicenter study, 147 ET were analyzed by reverse-transcriptase polymerase chain reaction (RT-PCR) and the molecular data statistically compared with all clinical data available. RESULTS: Most tumors expressed chimeric transcripts with fusion of EWS exon 7 to FLI1 exon 6 (75 of 147) (type I) or five (39 of 147) and EWS exon 10 to FLI1 exon 5 (eight of 147) or 6 (five of 147). In five cases, chimerism between EWS exon 9 and FLI1 exons 4 and EWS exon 7 and FLI1 exon 7 or 8 was observed. Fifteen cases of EWS-ERG rearrangement were identified. In 85 of these patients treated in the European Cooperative Ewing Sarcoma Studies, molecular results were analyzed in comparison to age, sex, tumor localization, tumor volume, and disease extension. No significant correlation between the various fusion types and these features were observed. Relapse-free survival (RFS) for the 31 patients with localized disease and fusion type I tended to be longer compared with the 24 patients with localized tumors bearing other chimeric transcripts (P = .04). CONCLUSION: Results suggest a possible advantage in PFS for patients with localized disease and fusion type I transcripts, although this will require prospective validation with a larger number of patients and longer follow-up periods.

Adolescent↗