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. Remarkably, the specific IGF1 receptor (IGF1R) antibody α-IR3 was functionally equivalent to IGF1. Short-term IGF1 incubation of cells stimulated survival kinase AKT and increased X-linked inhibitor of apoptosis (XIAP) protein which was associated with Apo2L/TRAIL resistance. In contrast, long-term IGF1 incubation resulted in repression of XIAP protein through ceramide (Cer) formation derived from de novo synthesis which was associated with Apo2L/TRAIL sensitization. Addition of ceramide synthase (CerS) inhibitor fumonisin B1 during long-term IGF1 treatment reduced XIAP repression and Apo2L/TRAIL-induced apoptosis. Noteworthy, the resistance to conventional chemotherapeutic agents was maintained in cells following chronic IGF1 treatment. Overall, the results suggest that chronic IGF1 treatment renders ESFT cells susceptible to Apo2L/TRAIL-induced apoptosis and may have important implications for the biology as well as the clinical management of refractory ESFT.

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. DESIGN: Computer prediction analysis of peptide binding, HLA-A2.1 stabilization assays, and induction of cytotoxic T-lymphocytes (CTL) in immunized HLA-A2.1 transgenic mice were used to assess the immunogenicity of native and modified peptides derived from the fusion region of EWS-FLI-1 type 1. CTL-killing of multiple ESFT family members in vitro, and control of established xenografts in vivo, was assessed. We also examined whether these peptides could induce human CTLs in vitro. RESULTS: EWS-FLI-1 type 1 peptides were unable to stabilize cell surface HLA-A2.1 and induced weak CTL activity against Ewing sarcoma cells. In contrast, peptides with modified anchor residues induced potent CTL killing of Ewing sarcoma cells presenting endogenous (native) peptides. The adoptive transfer of CTL specific for the modified peptide YLNPSVDSV resulted in enhanced survival of mice with established Ewing sarcoma xenografts. YLNPSVDSV-specific CTL displayed potent killing of multiple ESFT types in vitro: Ewing sarcoma, pPNET, Askin's Tumor, and Biphenotypic sarcoma. Stimulation of human peripheral blood mononuclear cells with YLNPSVDSV peptide resulted in potent CTL-killing. CONCLUSIONS: These data show that YLNPSVDSV peptide is a promising antigen for ESFT immunotherapy and warrants further clinical development.

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. Furthermore, EFT cells strongly expressing CD57 (HNK-1), a surface marker for migrating and proliferating neural crest cells, are more tumorigenic than cells with low expression of CD57, possibly mediated in part by enhanced adhesion and invasion. We contribute to the controversy about the cellular origin of EFT by clonal analysis, showing that EFT cells can differentiate similar to neural crest cells. These data increase our knowledge about the pathogenesis and heterogeneity of EFT.

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. METHODS: Synovial fibroblasts were isolated from patients with RA by enzymatic digestion and cultured under standard conditions. Cell cycle analysis was performed using flow cytometry and staining with propidium iodide. RASFs were synchronised or arrested in various phases of the cell cycle with 0.5 mM hydroxyurea or 2.5 microg/ml nocodazol and with foetal calf serum-free insulin-transferrin-sodium selenite supplemented medium. Apoptosis was induced by stimulation with 100 ng/ml FasL or 100 ng/ml TRAIL over 18 hours. The apoptotic response was measured using the Apo-ONE Homogenous Caspase-3/7 Assay (Promega GmbH, Mannheim, Germany) and the Cell Death Detection (ELISAPlus) (enzyme-linked immunosorbent assay) (Roche Diagnostics GmbH, Mannheim, Germany). Staurosporin-treated cells (1 microg/ml) served as a positive control. Expression of Fas and TRAIL receptors (TRAILR1-4) was determined by fluorescence-activated cell sorting analysis. RESULTS: Freshly isolated RASFs showed only low proliferation in vitro, and the rate decreased further over time, particularly when RASFs became confluent. RASFs expressed Fas, TRAIL receptor-1, and TRAIL receptor-2, and the expression levels were independent of the cell cycle. However, the proliferation rate significantly influenced the susceptibility to FasL- and TRAIL-induced apoptosis. Specifically, proliferating RASFs were less sensitive to FasL- and TRAIL-induced apoptosis than RASFs with a decreased proliferation rate. Furthermore, RASFs that were synchronised in S phase or G2/M phase were less sensitive to TRAIL-induced apoptosis than synchronised RASFs in G0/G1 phase. CONCLUSIONS: Our data indicate that the susceptibility of RASFs to FasL- and TRAIL-induced apoptosis depends on the cell cycle. These results may explain some conflicting data on the ability of RASFs to undergo FasL- and TRAIL-mediated cell death and suggest that strategies to sensitise RASFs to apoptosis may include the targeting of cell cycle-regulating genes.

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. We applied Affymetrix Human Genome U95Av2 oligonucleotide microarrays representing approximately 12,000 genes to generate the expression profiles of the CCSST cell lines GG-62, DTC-1, KAO, MST2, MST3, and Su-CC-S1 in comparison with 8 neuroblastoma, 7 Ewing tumor, and 6 osteosarcoma cell lines. Subsequent hierarchical clustering of microarray data clearly separated all four of the tumor types from each other and identified differentially expressed transcripts, which are characteristically up-regulated in CCSST. Statistical analysis revealed a group of 331 probe sets, representing approximately 300 significant (P < 0.001) differentially regulated genes, which clearly discriminated between the CCSST and other tumor samples. Besides genes that were already known to be highly expressed in CCSST, like S100A11 (S100 protein) or MITF (microphthalmia-associated transcription factor), this group shows an obvious portion of genes that are involved in cyclic AMP response or regulation, in pigmentation processes, or in neuronal structure and signaling. Comparison with other expression profile analyses on neuroectodermal childhood tumors confirms the high robustness of this strategy to characterize tumor entities based on their gene expression. We found the avian erythroblastic leukemia viral oncogene homologue 3 (ERBB3) to be one of the most dramatically up-regulated genes in CCSST. Quantitative real-time PCR and Northern blot analysis verified the mRNA abundance and confirmed the absence of the inhibitory transcript variant of this gene. The protein product of the member of the epidermal growth factor receptor family ERBB3 could be shown to be highly present in all of the CCSST cell lines investigated, as well as in 18 of 20 primary tumor biopsies. In conclusion, our data demonstrate new aspects of the phenotype and the biological behavior of CCSST and reveal ERBB3 to be a useful diagnostic marker.

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. Sensitisation of OS cells and hOBs to TRAIL/Apo2L did not correlate with a compatible change in the gene expression profile of the receptors for TRAIL/Apo2L determined by quantitative real-time RT-PCR. Also, sensitisation of the TRAIL/Apo2L death pathway did not rely entirely on the chemotherapy-induced, caspase-dependent cytotoxicity. Further, chemotherapy did not cause a compatible change in expression levels of proteins such as Bcl-2, Bcl-x(L), Bax, cIAP2, XIAP and survivin. However, ActD, DOX and CDDP downregulated expression of cFLIP in OS cells as well as expression of p21 in normal hOBs. Interestingly, while VP16 also extinguished cFLIP in OS cells, which were sensitised for TRAIL/Apo2L by VP16, VP16 induced cFLIP and enhanced p21 levels in normal hOBs, which remained refractory to VP16 plus TRAIL/Apo2L. Together, our data reveal that TRAIL/Apo2L combined with certain chemotherapeutic drugs is toxic to bone tumour and normal human cells and suggest that cotreatment with TRAIL/Apo2L and VP16 provides an attractive approach for selective killing of tumour cells while leaving unaffected 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. After a 72-h incubation period with 50 microM pamidronate, cell numbers were reduced by up to 80%, whereas the monophosphonate analog 3-aminopropyl phosphonate had no effect at concentrations up to 2 mM. Clodronate reduced cell viability by maximally 40% at 1 mM. These data provide the first evidence for a direct growth-inhibitory effect of pamidronate on Ewing's sarcoma cells. Hence, pamidronate definitely merits a more thorough exploration into its potential use in the therapy of patients with Ewing's sarcoma.

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. METHODS AND MATERIALS: Twelve different ES/pPNET cell lines were investigated in vitro and (after establishment as tumor xenografts in athymic nude mice) in vivo for their TNF-alpha mRNA expression (real-time quantitative reverse transcriptase polymerase chain reaction) and TNF-alpha protein production (in vitro: enhanced amplified sensitivity immunoassay; in vivo: immunohistochemistry) after exposure to different irradiation doses (2, 5, 10, 20, 30, or 40 Gy) and after different time intervals (1, 3, 6, 12, 24, 48, or 72 h after irradiation). The bioactivity of the TNF-alpha protein was evaluated in chromogenic cytotoxicity and neutralization assays. RESULTS: Nine out of 12 ES/pPNET cell lines express constitutively significant quantities of bioactive TNF-alpha in vitro. ES/pPNET cells originating from primary tumors secreted higher TNF-alpha levels than cells derived from metastatic lesions. In 5 of the 9 TNF-alpha-producing cell lines, TNF-alpha mRNA and protein levels were upregulated after irradiation exposure in a time- and dose-dependent manner. After establishment of the ES/pPNET cell lines in athymic nude mice, the radiation-induced TNF-alpha release could be demonstrated also in the xenograft tumors in vivo (analogous to the in vitro experiments). Using the same methods for quantitative analysis, it was determined that the TNF-alpha expression of the radiation-responsive tumor cells was up to 2000-fold higher compared to the maximal radiation-induced TNF-alpha release in normal lung tissue measured during the pneumonic phase. CONCLUSION: Certain ES/pPNET cell lines produce extremely large quantities of bioactive TNF-alpha after radiation exposure in a time- and dose-dependent manner. Radiation-induced TNF-alpha production of tumor cells may be of paramount importance in respect to not only tumor behavior, but also to potential damage to normal tissue and the clinical status of the host.

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. The dose-dependent adhesion to TSP-1 was inhibited by anti-integrin antibodies directed against the alpha 4 or beta 1 subunit, but not by control antibodies against other integrins. To localize the potential alpha 4 beta 1-binding site within the TSP-1 molecule, the protein was subjected to limited proteolysis with chymotrypsin in the absence of calcium. The stable 70-kDa core fragment produced under these conditions promoted alpha 4 beta 1-dependent osteosarcoma cell adhesion in a manner similar to that of the intact protein. Moreover adhesion experiments with neutralizing antibodies revealed that the adhesion was totally dependent on the alpha 4 beta 1 interaction. Further blocking experiments with potential inhibitory peptides revealed that the alpha 4 beta 1-mediated adhesion was not influenced by peptides containing the RGD sequence. Attachment to the 70-kDa fragment was strongly inhibited by the CS-1 peptide, which represents the most active recognition domain for alpha 4 beta 1 integrin in fibronectin. The present data provide evidence that TSP-1 contains an alpha 4 beta 1 integrin-binding site within the 70-kDa core region.

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. An apparent Michaelis constant K(m) = 116.2 +/- 18.9 microM and a maximum transport velocity V(max) = 191.6 +/- 13.9 pmol x (10(6) cells)(-1) x min(-1) were calculated for the sodium-independent transport. These results were compared with those determined in two malignantly transformed extracranial cell lines, the human Ewing's sarcoma cell lines VH-64 and CADO-ES-1.

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). We applied Affymetrix human cancer G110 arrays to compare the gene expression patterns of GG-62 and other cell lines derived from small blue round cell tumors of childhood. Hierarchical clustering of 463 differentially expressed genes distinguished GG-62 from the ETs, as well as the neuroblastomas, and revealed a cluster of 36 upregulated genes. Several of these genes are involved in signal transduction pathways that may be critical for maintaining cell transformation; some examples are avian erythroblastic leukemia viral oncogene homolog 3 ( ERBB3), neuregulin 1 ( NRG1), fibroblast growth factor 9 ( FGF9), and fibroblast growth factor receptor-1 ( FGFR1). Furthermore, genes near the chromosome-12q13 breakpoint exhibited increased expression of GG-62 including ERBB3, NR4A1 (nuclear receptor subfamily 4, group A, member 1), cyclin-dependent kinase 2 ( CDK2), and alpha 5 integrin ( ITGA5). Altogether our findings demonstrate the MMSP derivation of GG-62 and may shed light on the mechanisms of tumorigenesis in this rare disease.

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. Whereas ET lines demonstrated increased expression of microtubule-associated protein tau (MAPT), protein phosphatase 1 regulatory subunit 1A (PPP1R1A), NIMA (never in mitosis gene a)-related kinase 2 (NEK2), and cyclin D1 (CCND1), neuroblastoma samples exhibited high expression of wingless-type mouse mammary tumor virus integration site family member 11 (WNT11), Drosophila frizzled homolog 2 (FZD2), and adenomatous polyposis coli (APC) which are involved in regulating free beta-catenin levels. These genes likely maintain tumor-specific characteristics and participate in key downstream regulatory mechanisms. We also correlated the expression levels of up-regulated genes in ETs with their chromosomal localization and compared these data to the comparative genomic hybridization profiles of the cell lines. We demonstrate that gains of genetic material contribute essentially to differential gene expression.