Morphological differentiation of a murine neuroblastoma clone in monolayer culture induced by dexamethasone

Dexamethasone induces morphological differentiation in murine neuroblastoma cells in culture. The percentage of differentiated cells depends on the concentration of dexamethasone in the medium and duration of treatment. After drug removal (with or without replating), many cells maintain their differentiated phenotype. Dexamethasone-treated cells have larger soma and contain more protein. Dexamethasone also produces a concentration-dependent inhibition of population growth. Growth inhibition is complete by 2 days following treatment with dexamethasone 50 micrograms/ml. Complete growth inhibition is achieved prior to the complete expression of morphological differentiation. Androstenedione, testosterone, and 17-beta-estradiol--all steroids without glucocorticoid activity--have no differentiating effect, although they inhibit growth or cause cell death at higher concentrations.     

Reversal of snake neurotoxin binding to mammalian acetylcholine receptor by specific antiserum

Snake curaremimetic toxins are known to bind to the nicotinic acetylcholine receptor (AcChoR) [Changeux et al. (1970) Proc. Natl Acad. Sci. USA, 67, 1241-1247], thus blocking neuromuscular transmission, and producing respiratory failure in mammals. In the present paper we show that the toxic effects of Naja nigricollis toxin alpha to mammals can be efficiently reversed by toxin-alpha-specific antibodies. In vivo we observed that return to normal breathing in toxin-alpha-intoxicated and ventilated rats was 12 times faster after injection of specific antiserum or monoclonal antibody (M-alpha 1) as compared with control animals. Ex vivo we observed that return to normal contraction of a toxin-alpha-blocked phrenic nerve-hemidiaphragm preparation was 14 times more rapid after treatment with specific antiserum than after washings. In vitro we observed that antibodies accelerated the reversal of binding of [3H]toxin alpha to AcChoR prepared from rat diaphragm. The observation made in vitro furthermore indicates that antibodies are capable of destabilizing the [3H]toxin-AcChoR complex. A similar destabilization phenomenon occurs also in vivo, as inferred from measurements of receptor occupancy by [3H]toxin alpha in diaphragm of anaesthetized rats in the presence or absence of antibodies. The property of antibodies to reverse neurotoxin binding to AcChoR may be considered as a critical test for evaluation of the quality of a neurotoxin-specific antisera.     

Determination of population balances in a mixed culture by specific cell adhesion

Summary Specific cell adhesion can be used to monitor the population balance in a mixed culture. A mixture of twoEscherichia coli strains were separated and monitored based on their different expression of thelamB protein. The results are compared to those obtained by differential plate counts.     

Proteasome inhibition to maximize the apoptotic potential of cytokine therapy for murine neuroblastoma tumors

Human neuroblastomas possess several mechanisms of self-defense that may confer an ability to resist apoptosis and contribute to the observed difficulty in treating these tumors in the clinical setting. These molecular alterations may include defects in proapoptotic genes as well as the overexpression of prosurvival factors, such as Akt among others. As a key regulator of the turnover of proteins that modulate the cell cycle and mechanisms of apoptosis, the proteasome could serve as an important target for the treatment of neuroblastoma. The present studies provide the first evidence that bortezomib, a newly approved inhibitor of proteasome function, inhibits phosphorylation of Akt, induces the translocation of proapoptotic Bid, and potently enhances the apoptosis of murine neuroblastoma tumor cells in vitro. Furthermore, in that inhibitors of the Akt pathway can sensitize otherwise resistant TBJ/Neuro-2a cells to apoptosis induced by IFN-gamma plus TNF-alpha, we hypothesized that bortezomib also could sensitize these cells to IFN-gamma plus TNF-alpha. We demonstrate for the first time that bortezomib not only up-regulates the expression of receptors for IFN-gamma and TNF-alpha on both TBJ neuroblastoma and EOMA endothelial cell lines, but also markedly enhances the sensitivity of these cells to apoptosis induced by IFN-gamma plus TNF-alpha in vitro. Furthermore, bortezomib enhances the in vivo antitumor efficacy of IFN-gamma/TNF-alpha-inducing cytokines, including both IL-2 and IL-12 in mice bearing well-established primary and/or metastatic TBJ neuroblastoma tumors. Collectively, these studies suggest that bortezomib could be used therapeutically to enhance the proapoptotic and overall antitumor activity of systemic cytokine therapy in children with advanced neuroblastoma.     

Modification of glutamate- effects on neuroblastoma cells in culture by heavy metals

Sodium L-glutamate inhibited the growth (due to inhibition of cell division and cell death) of mouse neuroblastoma (NB) cells in culture in a dose dependent fashion. The sensitivity of adrenergic (NBA₂₍₁₎) and cholinergic (NBE^?) clones to L-glutamate was similar. Sodium D-glutamate, L-aspartate, α-ketoglutarate, glutamine, γ-aminobutyric acid and carbachol did not inhibit the growth of NB cells. Methylmercuric chloride (CH₃HgCl), inorganic mercury (HgCl₂), manganese chloride (MnCl₂) and lead tri-butyl acetate, by themselves inhibited the growth of NB cells in culture to a varying degree ranging from 41% to 49%. However, the combination of glutamate with CH₃HgCl, HgCl₂ and MnCl₂, produced a synergestic effect on growth inhibition of NB cells in culture. The combination of glutamate with lead tri-butyl acetate produced only an additive effect. Sodium kainate neither inhibited the growth nor potentiated the growth inhibitory effect of L-glutamate on NB cells. Neuroblastoma cells contained high levels of receptors for glutamate but not for kainate. These results show that neuroblastoma culture may be a useful model to study the mechanisms of glutamate effects and their modification by various agents.     

Protein structural effects of agonist binding to the nicotinic acetylcholine receptor.

The effects on the protein structure produced by binding of cholinergic agonists to purified acetylcholine receptor (AcChR) reconstituted into lipid vesicles, has been studied by Fourier-transform infrared spectroscopy and differential scanning calorimetry. Spectral changes in the conformationally sensitive amide I infrared band indicates that the exposure of the AcChR to the agonist carbamylcholine, under conditions which drive the AcChR into the desensitized state, produces alterations in the protein secondary structure. Quantitative estimation of these agonist-induced alterations by band-fitting analysis of the amide I spectral band reveals no appreciable changes in the percent of alpha-helix, but a decrease in beta-sheet structure, concomitant with an increase in less ordered structures. Additionally, agonist binding results in a concentration-dependent increase in the protein thermal stability, as indicated by the temperature dependence of the protein infrared spectrum and by calorimetric analysis, which further suggest that AcChR desensitization induced by the cholinergic agonist implies significant rearrangements in the protein structure.     

Cellular immune response to acetylcholine receptors in murine experimental autoimmune myasthenia gravis: inhibition with monoclonal anti-I-A antibodies

Gene(s) at the I-A subregion of the murine major histocompatibility complex influence susceptibility to experimental autoimmune myasthenia gravis. C57Bl/6 mice immunized with acetylcholine receptors (AChR) in complete Freund's adjuvant demonstrated cellular and humoral immune responses to AChR. They developed muscle weakness characteristic of myasthenia gravis and demonstrated a reduction in the muscle AChR content. The kinetics of AChR-specific lymphocyte proliferation generally correlate with anti-AChR antibody response. AChR-specific lymphocyte proliferation was also observed in C57Bl/6 splenocytes after secondary immunization with AChR. The in vitro cellular reactivity to AChR in experimental autoimmune myasthenia gravis (EAMG) mice (C57Bl/6) was suppressed by monoclonal anti-I-Ab antibodies directed against private (Ia20) or public (Ia8) specificities, suggesting a critical role for these Ia determinants in the cellular immune response to AChR in murine EAMG.     

Caffeine inhibits the binding of dimethylbenz(a)anthracene to murine epidermal cells DNA in culture.

The binding of dimethylbenz(α)anthracene to DNA of murine cells in culture is inhibited by caffeine and to a lesser extent by theophylline. However, other chemical analogs of caffeine such as theobromine, xanthine, hypoxanthine, and uric acid do not affect this process. Thus, the antitumorigenic effect of caffeine may be related to its ability to inhibit the binding of active metabolites of carcinogens to cellular DNA.     

Organization of ligand binding sites at the acetylcholine receptor: a study with monoclonal antibodies

We have studied 20 monoclonal antibodies directed against both the solubilized and the membrane-bound receptor from Torpedo marmorata. We find the following: (i) Six of the antibodies compete with cholinergic ligands for receptor binding and, hence, are directed against the ligand binding regions. (ii) Of these six antibodies, two cross-react with receptor from Electrophorus electricus, rat myotubes, and chicken sympathetic ganglia. These two antibodies therefore define a preserved structure within the ligand binding regions. The other four antibodies bind to structures not common between the receptor preparations tested. (iii) From competition binding studies using internally 3H-labeled antibodies, nine nonoverlapping antigenic regions were defined at the surface of the receptor. Three of these regions overlap with the ligand binding regions. Since two of these three regions do not overlap with each other, two structurally distinct ligand binding regions must exist at the receptor. (iv) From competition binding studies with representative cholinergic ligands, the antibodies directed against the ligand binding regions can be subdivided into three groups: one group competes with all ligands tested; the second group competes with all ligands except the bismethonium compounds; the third group competes with all ligands except the bismethonium compounds and tubocurarine. The results are summarized in a model of the organization of ligand binding sites at the receptor: There are two ligand binding regions differing in their antigenic properties. Furthermore, either there exists separate sites for distinct groups of ligands within each of these binding regions or some ligands produce conformational changes of the receptor that reversibly abolish some antigenic sites. In any case, the cholinergic ligands must interact with the receptor by more and/or other structural determinants than are provided by the structure of acetylcholine.     

Radioautography of binding of tritiated diprenorphine to opiate receptors in the rat

Radioautography of tritiated diprenorphine in rat brain indicates anatomic distribution of receptors with a greater degree of precision than is possible using dissection techniques. The results of this study largely confirm those of others but indicate some differences in receptor distribution in the thalamus. Differential receptor binding in the periaquaductal gray matter with the highest counts lying laterally is an original observation.     

Tracer and maximal specific binding of tritiated spiperone or N-n-propylnorapomorphine to quantify dopamine receptors in rat brain regions in vivo

Specific tracer and maximal specific binding (Bmax) were determined in rat brain regions from radioactivity accumulation after intravenous administration of 3H N-n-propylnorapomorphine (NPA) or 3H spiperone at various specific activities. With NPA the highest Bmax-values (expressed in pmol.g-1 tissue) were found in the striatum (26 pmol.g-1) nucleus accumbens (about 27 pmol.g-1) and the olfactory tubercle (11 pmol.g-1). Saturable NPA binding was also found in the amygdaloid complex, medulla oblongata and inferior colliculi, but not in the frontal cortex. Bmax values for spiperone were high in the striatum (73 pmol.g-1), the nucleus accumbens (48 pmol.g-1), the olfactory tubercle (34 pmol.g-1) and the frontal cortex (18 pmol.g-1). A similar order was found for the tracer contents in these regions. There was no linear relationship between these contents and Bmax values. The possible implications of these findings and usefulness of NPA for brain imaging are discussed.     

Estrogen effects in the myocardium: inhibition of NF-kappaB DNA binding by estrogen receptor-alpha and -beta

We have previously shown that estrogen effects in the heart include direct hormone effects on the myocardium. In a recent study we found that one beneficial effect of estradiol on the myocardium is the inhibition of apoptosis in cardiac myocytes. This effect was associated with a reduction of NF-kappaB activity. In the present study we have analyzed the functional mechanism of NF-kappaB inhibition in the myocardium by estrogen receptors-alpha and -beta. Despite the previous finding that 17-beta-estradiol (10 nM) inhibited the staurosporine-induced binding of p65/p50 NF-kappaB complexes to their cognate DNA elements in cultured rat cardiac myocytes, myocyte extracts showed no change in expression or cellular localization of p65, p50, and IkappaB upon staurosporine or estradiol treatment. Addition of either estrogen receptor-alpha or estrogen receptor-beta as recombinant protein was sufficient to inhibit staurosporine-dependent p65/p50 DNA binding in cardiac myocytes. 17-beta-Estradiol inhibits staurosporine-induced p65/p50 DNA binding associated with apoptotic cell death of cardiac myocytes via estrogen receptors-alpha and -beta. This is not associated with changes in p65, p50 and IkappaB expression or subcellular localization. Thus, inhibition of NF-kappaB activity by estrogenic compounds might inhibit NF-kappaB dependent gene expression such as pro-inflammatory cytokines in the myocardium.     

Growth inhibition of murine mammary carcinoma by monoclonal IgE antibodies specific for the mammary tumor virus

Summary Two IgE-producing hybridomas were established from spleen cells of Balb/c mice, which had been immunized with mouse mammary tumor virus (MMTV). These IgE monoclonal antibodies (mAbs) reacted specifically with the major envelope glycoprotein (gp36) of MMTV, as established by the immunoblot assay and by passive cutaneous anaphylaxis. The effect of the IgE mAbs (produced by clone A8) on the growth of the MMTV-secreting mammary adenocarcinoma H2712 was investigated in syngeneic C3H/HeJ mice. The mice were inoculated s.c. with either 10⁵ (100 × LD₅₀) or 10⁶ (1000 × LD₅₀) tumor cells and received repeated i.p. injections of 25 µg anti-gp36 IgE mAbs at 4-day intervals for 8 weeks. This treatment prevented the development of subcutaneous tumors in 50% of the animals. Similar protection was observed when the tumor cells (10⁵/animal) were injected i.p. 4 days prior to the beginning of the i.p. treatment consisting of injections of 25 µg mAbs at 4-day intervals for 6 weeks. However, these mAbs did not protect C3H/HeJ mice against the MMTV-negative MA16/c carcinoma cells. Hence, these results support the view that IgE-mediated cytotoxic mechanisms may play an immunologically specific antitumor surveillance role and that laboratory-induced antitumor IgE mAbs have the potential of specific therapeutic agents for in vivo destruction of tumor cells.