Development of curative therapies for Ewing sarcomas by interdisciplinary cooperative groups in Europe.

Curative therapies for Ewing sarcoma have been developed within cooperative groups. Consecutive clinical trials have systematically assessed the impact and timing of local therapy and the activity of cytotoxic drugs and their combinations. They have led to an increase of long-term disease-free survival to around 70% in patients with localized disease.

A Novel Role of IGF1 in Apo2L/TRAIL-Mediated Apoptosis of Ewing Tumor Cells.

Insulin-like growth factor 1 (IGF1) reputedly opposes chemotoxicity in Ewing sarcoma family of tumor (ESFT) cells. However, the effect of IGF1 on apoptosis induced by apoptosis ligand 2 (Apo2L)/tumor necrosis factor (TNF-) related apoptosis-inducing ligand (TRAIL) remains to be established. We find that opposite to the partial survival effect of short-term IGF1 treatment, long-term IGF1 treatment amplified Apo2L/TRAIL-induced apoptosis in Apo2L/TRAIL-sensitive but not resistant ESFT cell lines.

EWS-FLI-1-targeted cytotoxic T-cell killing of multiple tumor types belonging to the Ewing sarcoma family of tumors.

Purpose: The Ewing sarcoma family of tumors (ESFT) comprises a group of aggressive, malignant bone, and soft tissue tumors that predominantly affect children and young adults. These tumors frequently share expression of the EWS-FLI-1 translocation, which is central to tumor survival but not present in healthy cells. In this study, we examined EWS-FLI-1 antigens for their capacity to induce immunity against a range of ESFT types.

Ewing's sarcoma cells with CD57-associated increase of tumorigenicity and with neural crest-like differentiation capacity.

The Ewing family of tumors (EFT) is an important group of pediatric malignancies with a guarded prognosis. Little is known about the heterogeneity of EFT cells, and the cellular origin of EFT is disputed. We now add evidence that EFT are heterogeneous by showing that EFT cells from spheres growing in serum-free medium are markedly more tumorigenic than adherently growing EFT cells.

Susceptibility of rheumatoid arthritis synovial fibroblasts to FasL- and TRAIL-induced apoptosis is cell cycle-dependent.

Introduction: The rheumatoid arthritis (RA) synovium is characterised by the presence of an aggressive population of activated synovial fibroblasts (RASFs) that are prominently involved in the destruction of articular cartilage and bone. Accumulating evidence suggests that RASFs are relatively resistant to Fas-ligand (FasL)-induced apoptosis, but the data concerning tumour necrosis factor-related apoptosis-inducing ligand (TRAIL) have been conflicting. Here, we hypothesise that the susceptibility of RASFs to receptor-mediated apoptosis depends on the proliferation status of these cells and therefore analysed the cell cycle dependency of FasL- and TRAIL-induced programmed cell death of RASFs in vitro.

PI3K/AKT is involved in mediating survival signals that rescue Ewing tumour cells from fibroblast growth factor 2-induced cell death.

While in vitro studies had shown that fibroblast growth factor 2 (FGF2) can induce cell death in Ewing tumours, it remained unclear how Ewing tumour cells survive in vivo within a FGF2-rich microenvironment. Serum- and integrin-mediated survival signals were, therefore, studied in adherent monolayer and anchorage-independent colony cell cultures. In a panel of Ewing tumour cell lines, either adhesion to collagen or exposure to serum alone only had a minor protective effect against FGF2.

Expression profiling of t(12;22) positive clear cell sarcoma of soft tissue cell lines reveals characteristic up-regulation of potential new marker genes including ERBB3.

Clear cell sarcoma of soft tissue (CCSST), also known as malignant melanoma of soft parts, represents a rare lesion of the musculoskeletal system usually affecting adolescents and young adults. CCSST is typified by a chromosomal t(12;22)(q13;q12) translocation resulting in a fusion between the Ewing sarcoma gene (EWSR1) and activating transcription factor 1 (ATF1), of which the activity in nontransformed cells is regulated by cyclic AMP. Our aim was to identify critical differentially expressed genes in CCSST tumor cells in comparison with other solid tumors affecting children and young adults to better understand signaling pathways regulating specific features of the development and progression of this tumor entity.

Selective and nonselective toxicity of TRAIL/Apo2L combined with chemotherapy in human bone tumour cells vs. normal human cells.

Although TRAIL/Apo2L preferably induces apoptosis in tumour cells without toxicity in normal cells, many tumour cell types display TRAIL/Apo2L resistance. Whether TRAIL/Apo2L in combination with chemotherapy may overcome TRAIL/Apo2L resistance while maintaining tumour selectivity remains to be determined. Here, we report that while ActD, DOX and CDDP sensitised both OS and Ewing's tumour cell lines and normal cells (hOBs, synovial cells, fibroblasts) to TRAIL/Apo2L-induced apoptosis, the combination of etoposide (VP16) and TRAIL/Apo2L was selectively active on tumour cells without affecting normal cells.

The bisphosphonate pamidronate is a potent inhibitor of Ewing's sarcoma cell growth in vitro.

The MTT assay was used to measure the effects of pamidronate, clodronate and mevastatin on the cell viability of Ewing's sarcoma cell lines 6647, CADO-ES-1, ES-2, ES-3, RD-ES, SK-ES-1, STA-ET-2.1 and VH-64. Treatment of these cells with pamidronate inhibited cell viability in a time- and dose-dependent manner.

Ewing's sarcoma and peripheral primitive neuroectodermal tumor cells produce large quantities of bioactive tumor necrosis factor-alpha (TNF-alpha) after radiation exposure.

Purpose: In the present study, we examined human Ewing's sarcoma (ES) and peripheral primitive neuroectodermal tumor (pPNET) cell lines that are able to produce TNF-alpha as part of the response to irradiation. Radiation-induced tumor cell production of TNF-alpha may enhance irradiation efficacy and improve the effect of local tumor irradiation. On the other hand, radiation-induced tumor cell production of TNF-alpha may adversely affect the normal tissue.

Chromosomal alterations in osteosarcoma cell lines revealed by comparative genomic hybridization and multicolor karyotyping.

We characterized the chromosomal alterations in eight osteosarcoma cell lines (OST, HOS, U-2 OS, ZK-58, MG-63, SJSA-1, Saos-2, and MNNG) by comparative genomic hybridization (CGH); gains and losses of DNA sequences were defined as chromosomal regions with a fluorescence ratio, wherein all of the 95% confidence interval was above 1.25 and below 0.75, respectively.

Adhesion of osteosarcoma cells to the 70-kDa core region of thrombospondin-1 is mediated by the alpha 4 beta 1 integrin.

Thrombospondin-1 (TSP-1) is an extracellular glycoprotein that is involved in a variety of physiological processes such as tumor cell adhesion, invasion, and metastasis. It has been hypothesized that TSP-1 provides an adhesive matrix for osteosarcoma cells. Here we present data showing that TSP-1 can promote cell substrate adhesion to U2OS and SAOS cells through the alpha 4 beta 1 integrin.

3-[123I]Iodo-L-alpha-methyl tyrosine transport into human fibroblasts and comparison with Ewing's sarcoma cells.

The cellular transport systems and the transport kinetics of [123I]IMT uptake into non-malignant extracranial cells were characterized for the first time. Human fibroblasts were chosen as non-malignant extracranial cells as they are found ubiquitous in the body. [123I]IMT is exclusively transported into fibroblasts via the sodium independent system L.

Characterization of the malignant melanoma of soft-parts cell line GG-62 by expression analysis using DNA microarrays.

GG-62 is a cell line previously thought to be derived from an atypical Ewing tumor (ET). Reverse-transcriptase polymerase chain reaction revealed an in-frame fusion between the Ewing sarcoma gene ( EWS) codon 325 and the activating transcription factor 1 gene ( ATF1) codon 65 which permits the production of chimeric EWS-ATF1 oncoproteins. We also identified the genomic breakpoint resulting from a reciprocal t(12;22)(q13;q12), which is the hallmark of malignant melanoma of soft parts (MMSP).

Expression analysis of pediatric solid tumor cell lines using oligonucleotide microarrays.

We identified patterns of differentially-expressed genes in cell lines derived from several pediatric solid tumors. Affymetrix Human Cancer G110 Arrays, carrying 1,700 cancer-associated genes, were applied to a panel of 11 cell lines originating from Ewing tumors (ETs), neuroblastomas, and malignant melanoma of soft parts. Hierarchical clustering clearly differentiated these 3 entities and revealed groups of 75, 102, and 36 gene probe-sets exhibiting tumor-type specific up-regulation in these cell lines, respectively.

Sensitization for death receptor- or drug-induced apoptosis by re-expression of caspase-8 through demethylation or gene transfer.

Resistance of tumors to treatment with cytotoxic drugs, irradiation or immunotherapy may be due to disrupted apoptosis programs. Here, we report in a variety of different tumor cells including Ewing tumor, neuroblastoma, malignant brain tumors and melanoma that caspase-8 expression acts as a key determinant of sensitivity for apoptosis induced by death-inducing ligands or cytotoxic drugs. In tumor cell lines resistant to TRAIL, anti-CD95 or TNFalpha, caspase-8 protein and mRNA expression was decreased or absent without caspase-8 gene loss.

Alternative lengthening of telomeres is associated with chromosomal instability in osteosarcomas.

Telomere maintenance is regarded as a key mechanism in overcoming cellular senescence in tumor cells and in most cases is achieved by the activation of telomerase. However there is at least one alternative mechanism of telomere lengthening (ALT) which is characterized by heterogeneous and elongated telomeres in the absence of telomerase activity (TA). We evaluated the prevalence of TA, gene expression of telomerase subunits and ALT in relation to telomere morphology and function in matrix producing bone tumors and in osteosarcoma cell lines and present evidence of a direct association of ALT with telomere dysfunction and chromosomal instability.

The bisphosphonate pamidronate is a potent inhibitor of human osteosarcoma cell growth in vitro.

Bisphosphonates (BPs), such as pamidronate and clodronate, are an important class of drugs for the treatment of bone diseases. It is widely recognized that they inhibit bone resorption by suppressing the action of osteoclasts through antagonizing the mevalonate pathway, thereby reducing osteolytic bone metastases derived from different cancers, i.e.

Characterization of 3-[123I]iodo-L-alpha-methyl tyrosine ([123I]IMT) transport into human Ewing's sarcoma cells in vitro.

3-[(123)I]Iodo-L-alpha-methyl tyrosine ([(123)I]IMT) scintigraphy of extracranial malignant tumors has been described, but little is known about the transport systems involved in [(123)I]IMT uptake into extracranial tumor cells. Here, the precise kinetics of [(123)I]IMT transport into human Ewing's sarcoma cells (VH-64) was determined. The apparent Michaelis constant was of high affinity value (K(m)=41.

Establishment of an in vivo model for pediatric Ewing tumors by transplantation into NOD/scid mice.

Ewing tumors are a clinically heterogeneous group of childhood sarcomas that represent a paradigm for understanding solid tumor biology, as they are the first group of sarcomas for which a chromosome translocation has been characterized at the molecular level. However, the biologic organization of the tumor, especially the processes that govern proliferation, differentiation, and metastasis of primitive tumor stem cells is poorly understood. Therefore, to develop a biologically relevant in vivo model, five different Ewing tumor cell lines and primary tumor cells from three patients were transplanted into immune-deficient mice via intravenous injection.

Apoptotic responsiveness of the Ewing's sarcoma family of tumours to tumour necrosis factor-related apoptosis-inducing ligand (TRAIL).

We investigated the cytotoxic responsiveness of 40 cell lines derived from representatives of the Ewing's sarcoma family of tumours (ESFT), i.e., Ewing's sarcoma (ES), peripheral primitive neuroectodermal tumour (pPNET) and Askin tumour (AT), to tumour necrosis factor-related apoptosis-inducing ligand (TRAIL).

Cytotoxic effect of methotrexate and its solvent on osteosarcoma cells in vitro.

Bone tumours may recur locally even after wide surgical excision and systemic chemotherapy. Local control of growth may be accomplished by the addition of cytostatic drugs such as methotrexate (MTX) to bone cement used to fill the defect after surgery and to stabilise the reconstructive prosthesis. We have studied the elution kinetics of MTX and its solvent N-methyl-pyrrolidone (NMP) from bone cement and their biological activities in five cell lines of osteosarcoma and in osteoblasts, and compared them with the effects of the parent compounds alone and in combination.