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. Translational research in ES remains an area in which interdisciplinary and international cooperation is essential for future progress. This article reviews current state-of-the art therapy, with a focus on trials performed in Europe, and summarizes novel strategies to further advance both the cure rates and quality of survival.

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. Methylation-specific PCR revealed hypermethylation of caspase-8 regulatory sequences in cells with impaired caspase-8 expression. Treatment with the demethylation agent 5-Aza-2'-deoxycytidine (5-dAzaC) reversed hypermethylation of caspase-8 resulting in restoration of caspase-8 expression and recruitment and activation of caspase-8 at the CD95 DISC upon receptor cross-linking thereby sensitizing for death receptor-, and importantly, also for drug-induced apoptosis. Inhibition of caspase-8 activity also inhibited apoptosis sensitization by 5-dAzaC. Similar to demethylation, introduction of caspase-8 by gene transfer sensitized for apoptosis induction. Hypermethylation of caspase-8 was linked to reduced caspase-8 expression in different tumor cell lines in vitro and, most importantly, also in primary tumor samples. Thus, these findings indicate that re-expression of caspase-8, e.g. by demethylation or caspase-8 gene transfer, might be an effective strategy to restore sensitivity for chemotherapy- or death receptor-induced apoptosis in various tumors in vivo.

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. Telomere fluorescence in situ hybridization (T-FISH) in ALT cells revealed elongated and shortened telomeres, partly in unusual configurations and loci, dicentric marker chromosomes and signal-free chromosome ends. Free ends give rise to end-to-end associations and may induce breakage-fusion-bridge cycles resulting in an increased number of complex chromosomal rearrangements, as detected by multiplex-FISH (M-FISH). We propose that ALT cannot be seen as an equivalent to telomerase activity in telomere maintenance. Its association with telomere dysfunction and chromosomal instability may have major implications for tumor progression.

Vasoactive intestinal peptide receptor regulation of cAMP accumulation and glycogen hydrolysis in the human Ewing's sarcoma cell line WE-68.

This study describes functional characteristics of receptors for vasoactive intestinal peptide (VIP) on human Ewing's sarcoma WE-68 cells. These characteristics include 125I-VIP binding capacity, cellular cAMP generation, glycogen hydrolysis, and pharmacological specificity. Binding studies with 125I-VIP showed specific, saturable, binding sites for VIP in WE-68 cells. Scatchard analysis revealed the presence of a single class of high-affinity binding sites that exhibited a dissociation constant (Kd) of 90 pM and a maximal binding capacity (Bmax) of 24 fmol/mg of protein. VIP and VIP-related peptides competed for 125I-VIP binding in the following order of potency: human (h) VIP greater than human peptide with N-terminal histidine and C-terminal methionine (PHM) greater than chicken secretin much greater than porcine secretin. Glucagon and the C-terminal fragments VIP[10-28] and VIP[16-28] and the VIP analogue (D-Phe2)VIP did not inhibit 125I-VIP binding. Addition of hVIP to WE-68 cells provoked marked stimulation of cAMP accumulation, hVIP stimulated increases in cAMP content were rapid, concentration-dependent, and potentiated by 3-isobutyl-l-methylxanthine (IBMX). Half-maximal stimulation (EC50) occurred at 150 nM hVIP. The ability of hVIP and analogues to stimulate cAMP generation paralleled their potencies in displacing 125I-VIP binding. (D-Phe2)VIP, VIP[10-28], VIP[16-28], and (p-Cl-D-Phe6, Leu17)VIP, a putative VIP receptor antagonist, affected neither basal cAMP levels nor hVIP-induced cAMP accumulation. WE-68 cell responses to hVIP were desensitized by prior exposure to hVIP. Desensitization to hVIP did not modify the cAMP response to beta-adrenergic stimulation, and beta-adrenergic agonist desensitization did not modify responses to hVIP. hVIP also induced a time- and concentration-dependent hydrolysis of 3H-glycogen newly formed from 3H-glucose in WE-68 cultures. hVIP maximally decreased 3H-glycogen content by 36% with an EC50 value of about 8 nM. The order of potency of structurally related peptides of hVIP for stimulation of glycogenolysis correlated with their order of potency for inhibition of 125I-VIP binding. IBMX potentiated the glycogenolytic action of hVIP and PHM. The simultaneous presence of the calcium channel antagonist verapamil or the calcium ionophore A 23187 did not influence the glycogenolytic and cAMP stimulatory effects of hVIP. Collectively, these data indicate that Ewing's sarcoma (WE-68) cells are endowed with genuine VIP receptors which are coupled to the formation of cAMP that probably serves a second messenger role in stimulating glycogen hydrolysis in these cells in response to VIP.(ABSTRACT TRUNCATED AT 400 WORDS)

Functional characteristics of calcitonin gene-related peptide receptors in human Ewing's sarcoma WE-68 cells.

Calcitonin gene-related peptide (CGRP) receptor activity was studied in WE-68 human Ewing's sarcoma cells. 125I-human CGRP bound in a time-dependent, reversible and saturable manner. Scatchard plots were compatible with the presence of a homogenous population of CGRP receptors with high affinity (Kd = 15 pM, and Bmax = 1.9 fmol/mg protein). The potency order of unlabeled peptides in the presence of radioligand, was: human CGRP-II greater than human CGRP = chick CGRP greater than rat CGRP = rat [Tyr0]CGRP greater than human [Tyr0] CGRP much greater than salmon calcitonin (CT) greater than rat [Tyr0]CGRP-(28-37). Each peptide except CT and [Tyr0]CGRP-(28-37) stimulated cyclic AMP generation in a concentration-dependent manner, and the relative potencies paralleled their relative ability in inhibiting 125I-human CGRP binding. We conclude that WE-68 Ewing's sarcoma cells express genuine CGRP receptors which upon activation lead to stimulation of cyclic AMP formation

Neuropeptide Y inhibits vasoactive intestinal peptide- and dopamine-induced cyclic AMP formation in human Ewing's sarcoma WE-68 cells.

Neuropeptide Y (NPY) regulation of intracellular cyclic AMP accumulation was studied in human Ewing's sarcoma cell line, WE-68. NPY inhibited vasoactive intestinal peptide (VIP)- and dopamine-stimulated but not basal cyclic AMP formation. The peptide effect was rapid (less than 2 min), concentration-dependent with a half-maximal effective concentration (EC50) of 8 nM NPY, and maximal inhibition reaching 60-70% with 100 nM NPY. Prior exposure of WE-68 cells to pertussis toxin completely abolished the inhibitory action of NPY. It is concluded that NPY attenuates agonist-stimulated cyclic AMP formation in Ewing's sarcoma WE-68 cells, and may do so via the inhibitory guanine nucleotide regulatory protein of adenylate cyclase.

Forskolin inhibition of glucose transport in bone cell cultures through a cAMP-independent mechanism.

Studies are presented demonstrating inhibition of glucose transport by forskolin in human MG-63 osteosarcoma cells as well as osteoblast-like cells derived from normal human trabecular bone and chick calvaria. The cAMP-stimulators parathyroid hormone, prostaglandin E2, and isoproterenol did not influence glucose transport. Benzyl alcohol, a membrane lipid fluidity modulator, also provoked inhibition of the glucose uptake rate. Effects of forskolin and benzyl alcohol were not additive. It is suggested that cAMP is not a mediator of glucose transport in bone cells, and that forskolin inhibits glucose transport via a cAMP-independent mechanism.

Expression of functional very late antigen-alpha 1, -alpha 2, -alpha 3 and -alpha 6 integrins on Ewing's sarcoma and primitive peripheral neuroectodermal tumour cells and modulation by interferon-gamma and tumour necrosis factor-alpha.

Twelve different human primary and metastatic Ewing's sarcoma (ES) and primitive peripheral neuroectodermal tumour (pPNET) cell lines were examined by fluorocytometric analysis for the expression of alpha 1, alpha 2, alpha 3 and alpha 6 very late antigen (VLA) beta 1-integrins. VLA-alpha 1, was abundantly expressed on all typical ES cell lines and pPNET cell lines, while absent from atypical (large cell) ES cells. VLA-alpha 2 was displayed on some ES and pPNET cell lines. In two different pPNET cell lines, derived from the same patient, VLA-alpha 2 expression was considerably higher on primary cells compared with metastatic cells. VLA-alpha 3 was exclusively expressed on pPNET cell lines. Expression of VLA-alpha 6 was higher on metastatic than on primary ES and pPNET cells. Adhesion assays on purified extracellular matrix (ECM) proteins, using monospecific adhesion-blocking antibodies, disclosed VLA-1 (alpha 1 beta 1) on typical ES cells and pPNET cells, and VLA-2 (alpha 2 beta 1) on atypical ES cells, as dual collagen type IV (COIV)/laminin (LM) binding sites, and VLA-6 (alpha 6 beta 1) as a specific LM binding site. Treatment of typical ES cells and pPNET cells for up to 48 h with recombinant human interferon-gamma (rhIFN gamma) and tumour necrosis factor-alpha (rhTNF alpha) upregulated alpha 1 and beta 1 expression, concomitant with an increase in cell adhesion to COIV and LM. Alternatively, these cytokines downregulated the expression of alpha 2, alpha 6 and beta 1 on atypical ES cells, concomitant with a decrease in the adhesion to COIV and LM. In conclusion, these findings suggest that the difference in repertory of CO and LM integrin receptors on ES cells and pPNET cells reflects tumour status and degree of differentiation. Furthermore, our data indicate that IFN gamma- and TNF alpha-mediated alteration in the level of expression of distinct VLAs on ES and pPNET cells is correlated with changes in the adhesive behaviour of these tumour cells.

Monomorphic HLA class I-(non-A, non-B) expression on Ewing's tumor cell lines, modulation by TNF-alpha and IFN-gamma.

In this study, the expression of polymorphic and non-polymorphic MHC antigens in Ewing's tumor (ET) cells was examined by surface staining, Western blots and transcriptional analysis. Cell lines derived from Ewing's tumors largely lack polymorphic HLA class Ia antigens of both the HLA-A and the HLA-B loci but binding of monomorphic HLA antibodies indicates significant expression of HLA-C locus antigens and/or HLA class Ib molecules. HLA Ib molecules encoded by the HLA-E, -F or -G loci with a molecular mass of less than 44 kDa were not detected in lysates of either constitutive or TNF-alpha plus IFN-gamma treated ET cells. Two representative ET cell lines with either detectable HLA-A, -B antigens (A673) or absolutely non-detectable HLA-A, -B antigens (SK-ES-1) were further subjected to transcriptional analysis. A673 mRNA hybridized with HLA-A, -B, -C and HLA-E-specific probes in Northern blots. By contrast, mRNA specific for HLA-A, -B, -C was negative in SK-ES-1 but TNF-alpha plus IFN-gamma reconstituted HLA-A, -B, -C transcription in this cell line. HLA-E was transcribed in A673 but not in SK-ES-1. Combining mRNA and surface expression of HLA class Ia molecules results in a highly variable pattern of defective HLA class I expression in this type of neuroectodermal tumor. The involvement of the ET-specific fusion transcript EWS/Fli-1 in modulating the HLA-A and -B locus antigens is likely to occur by the upregulation of c-myc in these tumors. The exceptionally constant expression of HLA-C or some other non-A, non-B antigens (reactive with defined monoclonal antibodies) implies important consequences on tumor-cell resistance against specific CTL and NK activity in vivo.

Induction of glycogenolysis in cultured Ewing's sarcoma cells by dopamine and beta-adrenergic agonists.

This study describes hormonal regulation of glycogen metabolism in Ewing's sarcoma cells. 3H-Glycogen synthesized in cultured Ewing's sarcoma WE-68 cells from 3H-glucose was hydrolyzed in a concentration-dependent manner by various catecholamines. The order of potency for the glycogenolytic effects of catecholamines was isoproterenol greater than or equal to dopamine greater than norepinephrine greater than epinephrine. The concentrations giving half-maximal effectiveness (EC50) were about 2 x 10(-8) M, 3 x 10(-8) M, 8 x 10(-8) M, and 5 x 10(-7) M for isoproterenol, dopamine, norepinephrine, and epinephrine, respectively. Higher concentrations of each of the catecholamines were necessary to elicit EC50 stimulation of cyclic AMP production in Ewing's sarcoma cells. Glycogenolysis induced by dopamine was blocked by chlorpromazine, a dopamine D1-receptor antagonist, but not by haloperidol, a dopamine D2-receptor antagonist. The glycogenolytic action of norepinephrine was markedly reduced by propranolol, a beta-adrenoreceptor antagonist, and was not affected by yohimbine, an alpha-adrenoreceptor antagonist. In addition, chlorpromazine also antagonized the glycogenolytic response to norepinephrine. Dibutyryl cyclic AMP, 3-isobutyl-1-methylxanthine, and the diterpene forskolin were also found to induce 3H-glycogen hydrolysis. Our data indicate that catecholamines exert their glycogenolytic effects in Ewing's sarcoma cells by stimulation of cyclic AMP formation via beta-adrenergic receptors and dopamine D1-receptors.

Expression of functional Y1 receptors for neuropeptide Y in human Ewing's sarcoma cell lines.

In the human Ewing's sarcoma cell line WE-68, saturation analysis using 3H-labelled neuropeptide Y ([3H]NPY) as the radioligand disclosed a homogeneous population of binding sites with a dissociation constant (Kd) of 4.5 nM and maximal binding capacity (B(max)) of 712 fmol/mg cell protein. Besides the WE-68 cell line, ten other human Ewing's sarcoma cell lines (FM-62, HS-80, HT-78, HT-M1-78, NT-68, RM-82, RS-63, VH-64, WE-M1-68, WE-M2-68) were also found to display NPY receptors with Kd varying from 3.5 nM to 10.7 nM and B(max) = 247-3744 fmol/mg cell protein. NPY, its natural analogues and the Y1-receptor-specific peptide ligand [Leu31,Pro34]NPY inhibited [3H]NPY binding in the potency order: [Leu31,Pro34]NPY greater than or equal to human NPY greater than or equal to peptide YY (PYY) greater than salmon pancreatic polypeptide (PP) greater than human PP greater than porcine NPY13-36 much greater than NPY22-36. In the Ewing's sarcoma cell lines NPY provoked inhibition of forskolin-stimulated cyclic AMP formation by up to 98%. Pertussis toxin alleviated the cyclic-AMP-inhibitory response to NPY. In isolated Ewing's sarcoma plasma membranes pertussis toxin [32P]ADP-ribosylated a 41-kDa protein. The ability of NPY and analogues to inhibit cyclic AMP accumulation paralleled their potencies in displacing radioligand binding. By contrast, a cell line derived from an atypical form of Ewing's sarcoma did not express specific and functional NPY receptors. These results demonstrate that conventional Ewing's sarcoma cells possess Gi-protein-coupled NPY receptors of the Y1 type, which upon interaction with NPY, PYY, and PP mediate inhibition of cyclic AMP generation.

Type I and type II insulin-like growth factor receptors and their function in human Ewing's sarcoma cells.

Binding studies using recombinant human 125I-labelled insulin-like growth factor I ([125I]IGF-I) revealed IGF-I receptors in three Ewing's sarcoma cell lines with Kd ranging from 74 x 10(-12) M to 100 x 10(-12) M and Bmax = 36-63 fmol/mg cell protein. [125I]IGF-I binding was displaced by IGF-I, IGF-II and insulin with IC50 values of 1.5 nM, 6.3 nM and 0.7 microM respectively. Recombinant human [125I]IGF-II radioligand-binding assays in the cell lines disclosed specific binding sites for IGF-II with Kd = (110-175) x 10(-12) M and Bmax varying from 21 fmol/mg to 72 fmol/mg cell protein. Neither IGF-I nor insulin displaced [125I]IGF-II binding. IGF-I was found to increase basal glucose transport by maximally 1.5 times with EC50 = 0.9 nM IGF-I. The efficacy and potency of IGF-II on glucose uptake were comparable to those of IGF-I whereas insulin was ineffective. IGF-I and IGF-II also provoked stimulation of glycogen synthesis in Ewing's sarcoma cells. The maximal glycogenic response was reached at 0.01 microM IGF-I and 0.1 microM IGF-II, the EC50 value being approximately 1 nM IGF-I and 2 nM IGF-II. Insulin did not significantly influence glycogen formation. IGF-I and IGF-II but not insulin increased DNA synthesis in Ewing's sarcoma cells. The maximal mitogenic response was obtained with 10 nM IGF-I or IGF-II with an EC50 value of about 0.7 nM for both peptides. alpha-IR-3, a monoclonal antibody specific for the IGF type I receptor, effectively blocked IGF-I- and IGF-II-mediated metabolic responses. In conclusion, the data show that IGF-I and IGF-II induce rapid and long-term biological responses in Ewing's sarcoma cells exclusively through interaction with IGF type I receptors.

Interferon gamma and tumour necrosis factor alpha induce a synergistic antiproliferative response in human Ewing's sarcoma cells in vitro.

Three human cell lines derived from Ewing's sarcoma (RM-82, VH-64, and WE-68) were investigated to establish the influence of recombinant human interferon gamma (rhIFN gamma) and tumour necrosis factor alpha (rhTNF alpha) on cell proliferation and survival and to characterize IFN gamma and TNF alpha receptor expression. Incorporation of [3H]thymidine into cells was inhibited by rhIFN gamma after 24 h of incubation. Half-maximal inhibition was observed with 10-80 U/ml rhIFN gamma. A maximal effect (50%-70% inhibition of cell proliferation) was achieved by treatment of cells with 250 U/ml rhIFN gamma. The influence of rhTNF alpha on proliferation was found to differ among cell lines and varied with the concentration and the duration of exposure of cells to this cytokine. In WE-68 and VH-64 cells [3H]thymidine incorporation was not affected by rhTNF alpha up to 2000 U/ml after 96 h of incubation, whereas in RM-82 cells the incorporation was inhibited by 35% after 48 h of incubation with 100 U/ml rhTNF alpha. However, all cell lines showed a synergistic antiproliferative response to the combination of rhIFN gamma and rhTNF alpha after 24 h of incubation. The human recombinant cytokines interleukin(IL)-1 alpha, IL-1 beta, IL-2, IL-3, IL-4, IL-6 and granulocyte/macrophage-colony-stimulating factor, tested alone and in combination with rhIFN gamma and rhTNF alpha, had no influence on cell proliferation. Binding studies in the cell lines with 125I-rhIFN gamma revealed a dissociation constant (Kd) of 160-306 pM and approximately 8000-13,500 receptors/cell. Binding experiments with 125I-rhTNF alpha indicated 430-1250 receptors/cell with Kd ranging from 13 pM to 162 pM. These data indicate that, among various cytokines, only IFN and TNF alpha are capable of potently reducing Ewing's sarcoma cell growth in vitro. Our data suggest that IFN alone or in combination with TNF alpha may be useful in the design of novel strategies in Ewing's sarcoma therapy.

Regulation of the release of tumour necrosis factor (TNF)alpha and soluble TNF receptor by gamma irradiation and interferon gamma in Ewing's sarcoma/peripheral primitive neuroectodermal tumour cells.

This study analyses the production of tumour necrosis factor (TNF)alpha and soluble TNF receptor (sTNF-R) before and after exposure to gamma irradiation and interferon gamma (IFN gamma) in 12 cell lines derived from Ewing's sarcoma (ES)/peripheral primitive neuroectodermal tumours (pPNET). Supernatants from ES/pPNET cell cultures were tested in a TNF alpha-specific amplified enzyme-linked immunosorbent assay (ELISA), a bioassay, and sTNF-Rp55 and sTNF-Rp75 ELISA. The tumour cell lines released minimal amounts of TNF alpha, prominent amounts of sTNF-Rp55 (7/12 cell lines) and no sTNF-Rp75. Exposure to gamma irradiation (5 Gy) either induced (3/12) cell lines) or up-regulated (3/12 cell lines) TNF alpha release without changing sTNF-Rp55 and sTNF-Rp75 levels. Priming of cultures with recombinant human IFN gamma (rhIFN gamma) markedly enhanced TNF alpha secretion in the radiation-responsive cell lines and had no influence on sTNF-Rp55 and sTNF-Rp75 levels. rhIFN gamma affected the magnitude rather than the sensitivity of the radiation response. The TNF alpha secreted was bioactive, as shown by its cytotoxic effect of WEHI-164 cells, and neutralization of its activity by anti-TNF alpha monoclonal antibody. Herbimycin A (a tyrosine-specific protein kinase inhibitor) but not calphostin C (a protein kinase C inhibitor), H89 (a protein kinase A inhibitor), AA-COCF3 (a specific inhibitor of phospholipase A2) and MK-886 (a specific inhibitor of 5-lipoxygenase) abrogated gamma-irradiation-stimulated TNF alpha release. The antioxidants N-acetylcysteine, nordihydroguaiaretic acid and mepacrine dose-dependently inhibited gamma-irradiation-mediated TNF alpha production. Collectively our findings indicate that IFN gamma priming potentiates the secretion of bioactive TNF alpha by ES/pPNET cells in response to gamma irradiation without affecting sTNF-R release. The data suggest a requirement for protein tyrosine kinase activity and a role for reactive oxygen species in the gamma-irradiation-mediated intracellular signalling pathway leading to TNF alpha production.

Diagnostic value of the molecular genetic detection of the t(11;22) translocation in Ewing's tumours.

One consistent feature of the Ewing's tumour family is the presence of a balanced translocation involving band q12 and band q24 of chromosome 22 and chromosome 11. Recent cloning of the chromosome breakpoint regions of t(11;22)(q24;q12) Ewing's sarcoma translocation has revealed that the breakpoints were localized within the Ewing's sarcoma gene (EWS gene) on chromosome 22 and the Fli-1 gene on chromosome 11. Molecular genetic techniques can thus be applied to the detection of the t(11;22) translocation in Ewing's tumours. By reverse transcription and polymerase chain reaction technique (RT-PCR) 11 Ewing's tumour derived cell lines, 12 primary Ewing's tumours, and 11 tumours after treatment were analysed for the occurence of the t(11;22) translocation. Furthermore, blood and bone marrow samples from 5 patients were available for RT-PCR. In 78% of the cell lines and 91% of the primary Ewing's tumours the t(11;22) translocation was detectable by RT-PCR. In bone marrow samples from a Ewing's sarcoma patient presenting in relapse tumour cells were detected by molecular genetic analysis. Our results indicate that molecular genetic detection of the t(11;22) translocation is valuable in the differential diagnosis of small round cell tumours and will provide important information for the staging and prognosis of Ewing's tumour.

Calcitonin gene-related peptide (CGRP) receptors are linked to cyclic adenosine monophosphate production in SK-N-MC human neuroblastoma cells.

Calcitonin gene-related peptide (CGRP) stimulated cyclic adenosine monophosphate (cAMP) levels in SK-N-MC human neuroblastoma cells in a time- and concentration-dependent manner. The efficacy order for CGRPs was human alpha-CGRP = human beta-CGRP = chick CGRP greater than rat CGRP greater than human [Tyr0]CGRP. Calcitonin (CT) failed to influence cAMP production in SK-N-MC cells. [Tyr0]CGRP27-37 which by itself did not affect cAMP levels antagonized CGRP action. Saturation analysis using [125I]CGRP showed a homogeneous population of binding sites. CGRP but not CT, vasoactive intestinal peptide (VIP) and neuropeptide Y (NPY) inhibited radioligand binding. Our results provide evidence that human neuroblastoma SK-N-MC cells contain highly specific CGRP receptors which are positively coupled to cAMP generation.

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)=41.7+/-3.9 microM) and maximum transport velocitiy amounted to V(max)=20.7+/-0.6 nmol x mg protein(-1) x 10 min(-1). Inhibition experiments revealed the predominance of [(123)I]IMT uptake via sodium-independent system L.

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. Our findings show that MTX is released continuously over months at concentrations highly cytotoxic to osteosarcoma cells and suggest that the impregnated bone cement would be effective in the long term. Proliferating osteoblasts, however, were much less sensitive towards MTX. The dose-response relationship for NMP and experiments with MTX/NMP-mixtures show that the eluted concentrations of solvent are not toxic and do not influence the effects of MTX. We suggest that bone cement containing MTX dissolved in NMP releases the drug in a suitable and effective way and may be of value in the treatment of bone tumours.

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. However, both combined led to complete resistance to 5 ng ml(-1) FGF2 in three of four FGF2-sensitive cell lines (RD-ES, RM-82 and WE-68), and to an increased survival as compared to other culture conditions in TC-71 cells. Inhibition studies with LY294002 demonstrated that the serum signal is mediated via the phosphoinositide 3-OH kinase/AKT pathway. Thus, Ewing tumour cells escape FGF2-induced cell death by modulating FGF2 signalling. The tumour microenvironment provides the necessary survival signals by integrin-mediated adhesion and soluble serum factor(s). These survival signals warrant further investigation as a potential resistance mechanism to other apoptosis-inducing agents in vivo.

Gold(III) inhibits activated human osteosarcoma adenylate cyclase by an action at or near the catalytic component.

In human osteosarcoma membranes, gold(III) (Au(III)) inhibits prostaglandin E2- and isoproterenol-mediated stimulation of adenylate cyclase activity without affecting basal enzyme activity. Forskolin activation of adenylate cyclase is also blocked by Au(III) with ID50 of 1-2 microM. The inhibition by Au(III) is preserved in membranes prepared from pertussis toxin-treated cells. The inhibitory effect of Au(III) is additive to inhibition of adenylate cyclase by 2',5'-dideoxyadenosine. These data provide evidence that the action of Au(III) is at or near the catalytic moiety of the cyclase system and that Au(III) does not act via the guanine nucleotide-binding inhibitory component or the adenosine P-site inhibitory pathway.