Antiinflammatory effects of glucocorticoids in brain cells, independent of NF-kappa B.
(73/5127)
Glucocorticoids are potent antiinflammatory drugs. They inhibit the expression of proinflammatory cytokines and adhesion molecules. It has recently been proposed that the underlying basis to such inhibition is the induction of the protein I kappa B, which inhibits the transcription factor NF-kappa B. The latter is a key activator of the genes encoding cytokines and adhesion molecules. The present study shows that the synthetic glucocorticoid, dexamethasone, inhibits the induction of the proinflammatory cytokine IL-8 and the adhesion molecules VCAM-1 and ICAM-1 in human 1321N1 astrocytoma and SK.N.SH neuroblastoma cells. However, dexamethasone failed to induce I kappa B or inhibit activation of NF-kappa B by IL-1 in the two cell types. EMSA confirmed the identity of the activated NF-kappa B by demonstrating that an oligonucleotide, containing the wild-type NF-kappa B-binding motif, inhibited formation of the NF-kappa B-DNA complexes whereas a mutated form of the NF-kappa B-binding motif was ineffective. In addition, supershift analysis showed that the protein subunits p50 and p65 were prevalent components in the activated NF-kappa B complexes. The lack of effect of dexamethasone on the capacity of IL-1 to activate NF-kappa B correlated with its inability to induce I kappa B and the ability of IL-1 to cause degradation of I kappa B, even in the presence of dexamethasone. The results presented in this paper strongly suggest that glucocorticoids may exert antiinflammatory effects in cells of neural origin by a mechanism(s) independent of NF-kappa B. (+info)
Telomerase activity distinguishes between neuroblastomas with good and poor prognosis.
(74/5127)
BACKGROUND: Treatment of neuroblastoma has remained a major challenge in pediatric oncology because the assessment of the individual prognosis, particularly in disseminated disease is still obscure. Previous studies have correlated clinical outcome with activity levels of telomerase, a cellular reverse transcriptase which has been detected in the majority of human malignant tumors. PATIENTS AND METHODS: In this blind-trial study, a non-radioactive telomeric repeat amplification protocol (TRAP) with an internal telomerase-assay standard was used on an automated laser fluorescence sequencer for the detection and semiquantitative analysis of telomerase activity (TA) in 67 neuroblastomas of all clinical stages from the German Neuroblastoma Trial and 2 ganglioneuromas. TA levels were correlated with event-free and overall survival rates and established prognostic markers such as MYCN. RESULTS: TA was present in 14 of 69 (20%) samples, including 3 of 22 stage IVS, 8 of 14 stage IV, 1 of 10 stage III, 1 of 7 stage II and 1 of 14 stage I neuroblastomas and 0 of 2 ganglioneuromas. We found a strong statistical correlation between the presence of TA and poor clinical prognosis with regard to all tumor stages. Multivariate analysis revealed TA as an independent prognostic marker. In particular, the analysis of TA in IVS neuroblastomas distinguished two different prognostic groups. CONCLUSIONS: Our data suggest that TA is an independent prognostic marker in neuroblastoma which, in combination with other markers such as MYCN, may proof useful in assessing the individual patient's prognosis. (+info)
Human natural immunoglobulin M antibodies induce apoptosis of human neuroblastoma cells by binding to a Mr 260,000 antigen.
(75/5127)
Sera of healthy humans contain natural cytotoxic IgM antibodies that specifically recognize a Mr 260,000 antigen (NB-p260) on the surface of human neuroblastoma (NB) cells. Here we demonstrate that anti-NB IgM antibodies prepared from different healthy individuals induce, in all human NB cell lines analyzed thus far, typical morphological and biochemical features of apoptosis including nuclear fragmentation, chromatin condensation, and DNA fragmentation. Both the binding of human anti-NB IgM to NB cells and the induction of apoptosis could be inhibited by preincubation of NB cells with murine IgG raised against purified NB-p260. Furthermore, preincubation of human anti-NB IgM with purified NB-p260 immobilized onto a solid support abolished its ability to induce apoptosis in NB cells. Natural human anti-NB IgM failed to bind to and induce apoptosis in control tumor cell lines that lack expression of NB-p260. The anti-NB IgM-induced apoptotic response was also observed in vivo in xenografted human NB tumors. After a single i.v. injection of anti-NB IgM into nude rats bearing solid NB xenografts, many areas of pyknotic cells with fragmented nuclei were observed that stained positive using the terminal dUTP nick end labeling method. In conclusion, the data demonstrate that natural anti-NB IgM antibodies in the sera of healthy individuals are potent mediators of apoptotic cell death of NB cells both in vitro and in vivo. The NB-p260 antigen was identified as the apoptosis-inducing receptor for anti-NB IgM. Whereas natural anti-NB IgM and NB-p260 may be useful tools for immunotherapy of NB, their biological significance remains to be determined. (+info)
Neonatal neuroblastoma.
(76/5127)
Over nine years, 33 children with neonatal neuroblastoma were registered with the UKCCSG (United Kingdom Children's Cancer Study Group). Tumours of all stages were found, but stage 4S disease predominated. Five tumours were detected prenatally by ultrasonography. Treatment varied according to tumour stage. The overall survival of the group was 91%. Ten children have had long term complications as a result of their disease, usually as a result of spinal tumour involvement. The good overall prognosis in this age group is encouraging, but the poor neurological outcome of patients with intraspinal extension is of concern. (+info)
Monoclonal antibody to the delta opioid receptor acts as an agonist in dual regulation of adenylate cyclase in NG108-15 cells.
(77/5127)
Monoclonal antibodies generated against multiple antigenic peptides of the N-terminal sequence (3LVPSARAELQSSPLV17) of the cloned delta opioid receptor immunoprecipitated a 58 kDa protein from CHAPS-solubilized NG108-15 membranes. The immunoprecipitates bound [3H]DPDPE--but not [3H]DAMGO--with a Kd of 6.4 nM and a Bmax of 75 pM. Western blot analysis revealed a distinct band of 58 kDa. The antibodies inhibited basal and PGE1-stimulated cAMP levels, and mimicked the effect of agonists manifest in a compensatory increase in cAMP formation. The antibody will be potentially useful in the analysis of functional epitopes on the delta opioid receptor. (+info)
Induction of apoptosis by IFNgamma in human neuroblastoma cell lines through the CD95/CD95L autocrine circuit.
(78/5127)
The CD95 (APO-1/Fas) system can mediate apoptosis in immune cells as well as in tumour cells, where it may contribute to tumour immune-escape. On the other hand, its induction by anticancer drugs may lead to tumour reduction. Interferongamma (IFNgamma) increases the sensitivity of tumour cell lines to anti-CD95 antibody-mediated apoptosis. We describe induction of apoptosis by IFNgamma through the expression of CD95 and its ligand (CD95L) in human neuroblastoma cell lines. Neuroblastoma cells showed low constitutive expression of CD95 and CD95L. Subsequent to IFNgamma-modulated increase in CD95 and CD95L mRNA as well as protein levels, apoptosis was observed. Our results demonstrated that cytokine-mediated apoptosis was mediated through the activation of the CD95/CD95L autocrine circuit since: (i) cell death occurred following CD95/CD95L expression and correlated with CD95 and CD95L expression levels, (ii) failed to occur in a clone which weakly upregulated CD95 and lacked CD95L induction after IFNgamma stimulation, (iii) was at least partially inhibited by using blocking F(ab')2 anti-CD95 antibody fragments and the recombinant Fas-Fc protein, that prevented the interaction between CD95 and CD95L. The intracellular molecular mechanisms elicited by IFNgamma are clearly highly complex, with several signalling pathways being activated, including the CD95 system. These findings suggest that IFNgamma may have a significant potential in the therapy of neuroblastoma in vivo. (+info)
Separation of M-like current and ERG current in NG108-15 cells.
(79/5127)
Differentiated NG108-15 neuroblastoma x glioma hybrid cells were whole-cell voltage-clamped. Hyperpolarizing pulses, superimposed on a depolarized holding potential (-30 or -20 mV), elicited deactivation currents which consisted of two components, distinguishable by fitting with two exponential functions. Linopirdine [DuP 996, 3,3-bis(4-pyridinylmethyl)-1-phenylindolin-2-one), a neurotransmitter-release enhancer known as potent and selective blocker of the M-current of rat sympathetic neurons, in concentrations of 5 or 10 microM selectively inhibited the fast component (IC50 = 14.7 microM). The slow component was less sensitive to linopirdine (IC50>20 microM). The class III antiarrhythmics [(4-methylsulphonyl)amido]benzenesulphonamide (WAY-123.398) and 1-[2-(6-methyl-2-pyrydinil)ethyl]-4-(4-methylsulphonylaminobenz oyl) piperidine (E-4031), selective inhibitors of the inwardly rectifying ERG (ether-a-go-go-related gene) potassium channel, inhibited predominantly the slow component (IC50 = 38 nM for E-4031). The time constant of the WAY-123.398-sensitive current resembled the time constant of the slow component in size and voltage dependence. Inwardly rectifying ERG currents, recorded in K+ -rich bath at strongly negative pulse potentials, resembled the slow component of the deactivation current in their low sensitivity to linopirdine (28% inhibition at 50 microM). The size of the slow component varied greatly between cells. Accordingly, varied the effect of WAY-123.398 on deactivation current and holding current. RNA transcripts for the following members of the ether-a-go-go gene (EAG) K+ channel family were found in differentiated NG108-15 cells: ERG1, ERG2, EAGI, EAG-like (ELK)1, ELK2; ERG3 was only present in non-differentiated cells. In addition, RNA transcripts for KCNQ2 and KCNQ3 were found in differentiated and non-differentiated cells. We conclude that the fast component of the deactivation current is M-like current and the slow component is deactivating ERG current. The molecular correlates are probably KCNQ2/KCNQ3 and ERG1/ERG2, respectively. (+info)
Down-regulation of endothelial cell growth inhibitors by enhanced MYCN oncogene expression in human neuroblastoma cells.
(80/5127)
Recent evidence indicates that the genetic alterations of the multistage process of malignant transformation appear to activate tumor neovascularization by altering the balance between stimulators and inhibitors of angiogenesis. In the present study, we have attempted to define the effect of enhanced MYCN oncogene expression on the profile of endothelial cell growth modulators in neuroblastoma cells. We report here that conditioned medium of human neuroblastoma cells with normal MYCN expression contains three inhibitors of endothelial cell proliferation, which appear to be novel proteins as judged by their physicochemical, immunological and biological properties. All three inhibitors are diminished or become undetectable upon experimental increase of MYCN expression. Our results suggest that enhanced MYCN expression in human neuroblastoma cells alters the angiogenic balance by down-regulating endothelial cell growth inhibitors but leaving the expression of the stimulators unaffected. These data shed light on the molecular mechanisms linking the genetic changes of malignant transformation with initiation of tumor angiogenesis. Moreover, our observations might explain the poor prognosis of human neuroblastomas following MYCN oncogene amplification through initiation of angiogenesis and subsequent tumor growth and spread. (+info)