Antitumoural action of L-733,060 on neuroblastoma and glioma cell lines

We have performed an in vitro study of the growth-inhibitory capacity of the potent and long-acting NK1 receptor antagonist L-733,060, at concentrations ranging from 2.5 microM to 20 microM, against the neuroblastoma cell line SKN-BE(2) and 10 microM to 25 microM for glioma cell line GAMG. Coulter counter was used to determine viable cell numbers, followed by application of the tetrazolium compound [3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)2-(4-sulfophenyl)-2H-tetrazolium], inner salt colorimetric method to evaluate cell viability in this cytotoxicity assay. L-733,060 inhibited the growth of the two cell lines studied in a dose-dependent manner. The IC 50 values were 11.6 microM (30h) and 10.2 microM (72h) for SKN-BE(2); and 21.3 microM (48h) and 19.9 microM (96h) for GAMG. These findings indicate that the NK1 receptor antagonist L-733,060 acts as a broad-spectrum antitumoural agent. This new action, reported here for the first time, suggests that the NK1 receptor antagonist L-733,060 could be a promising therapeutic drug for the treatment of human neuroblastoma and human glioma.     

Phosphatase activity in cultured glioma and neuroblastoma cells

Acid phosphatase activity in human glioma cells (138 MG) and mouse neuroblastoma cells (C 1300) was associated with structures accumulating neutral red and acridine orange. Only neuroblastoma cells gave a significant positive histochemical reaction for alkaline phosphatase. Glioma and neuroblastoma cell homogenates exhibited maximal phosphatase activity at pH 5 as measured by spectrophotometer. The specific activity; μmoles phosphate released per hour/mg protein was 1.1 in glioma and 0.9 in neuroblastoma. At pH 8, glioma cells lacked activity whereas neuroblastoma cells showed another maximum. The acid phosphatase activity of both cell types was strongly inhibited by CuCl₂ (0.3 mM) and NaF (10 mM) and moderately by -tartaric acid (10 mM). cGMP (1 mM) stimulated the phosphatase activity of both cell lines. db-cAMP, in serum-free medium, induced characteristic morphological changes of the cells studied. This process was unaffected by CuCl₂, c-GMP and -tartaric acid. db-cAMP (1 mM) inhibited proliferation in both glioma and neuroblastoma cells during a 48 h incubation in serum-containing medium. This growth inhibition was associated with an increase in acid phosphatase activity of the glioma but not of the neuroblastoma cells.     

Effects of the 5-lipoxygenase inhibitors AA-863 and U-60,257 on human glioma cell lines

The effects of two specific 5-lipoxygenase inhibitors AA-863 and U-60,257 (piriprost) on the growth of two human glioma cell lines, U-343 MGa and U-251 MG were investigated. Both monolayer cultured cells and spheroids were studied. The results of the monolayer studies showed potent and dose dependent inhibitory effects on the proliferation of glioma cells (IC50/one week treatment/of AA-863: 9.0 microM, IC50 of U-60,257: 40.0 microM). The experiments made on the tumor spheroids suggested an inhibitory effect on proliferation and volume growth already at lower doses (AA-863: 0.4-2.0 microM, U-60,257: 1.0-5.0 microM), a dose range where effects were not found in monolayers. At higher doses (AA-863: 10.0-30.0 microM, U-60,257: 30.0-90.0 microM) the experiments with spheroids failed to demonstrate a further inhibitory effect on spheroid volume, probably attributed to phenomena such as swelling of cells, dissociation of spheroid structure and development of necrosis. The clearly dose dependent inhibitory effect on the proliferation of human glioma cells in monolayer culture and the inhibitory effects on spheroid growth with these specific inhibitors indicate a role for lipoxygenase products in the growth of gliomas.     

Effects of the PAF-analog and -antagonist CV-6209 on cultured human glioma cell lines

Cell lines of human glioma (U-343 MGa and U-251 MG) and human glia (U-533 CG) origin were cultured as monolayers and exposed to CV-6209, an alkyl-phospholipid analog and antagonist of platelet activating factor. This drug had very potent antiproliferative effects on the studied human glioma cell lines; IC50 was 0.9 microM after 48 h treatment and 0.2 microM after 2 weeks treatment. At these doses no growth inhibitory effect was noted on the normal glia cells. The effects on the glioma cells were reversible in the dose intervals, where cell proliferation, 3H-thymidine and 14C-methionine uptakes were greatly inhibited. The simultaneous administration of platelet activating factor [(R)PAF] did not influence the antiproliferative effects of CV-6209 on the cells cultured as monolayers. The structurally similar analog CV-3988 also had antiproliferative effects, although at 10 times higher concentration than CV-6209. Two other, structurally unrelated, PAF-antagonists (WEB-2086 and TCV-309) gave effects only at very high concentrations. The U-343 MGa cell line was also exposed to CV-6209 when growing as multicellular spheroids. The studies on the spheroid cultures also demonstrated good antitumoral effects with decreases of both the volume growth and the thymidine uptake. The simultaneous administration of (R)PAF reversed the inhibitory effect of CV-6209 on thymidine incorporation. This study demonstrates a strong antitumoral effect at low concentrations of CV-6209. The antiproliferative effects were probably primarily related to the ether-lipid structure and not to the PAF-antagonistic properties. The good antitumoral effect of CV-6209 on both monolayer and spheroid cultures and the possible PAF-antagonistic properties are discussed.     

Effects of salicylhydroxamic acid on the proliferation of Atriplex and murine neuroblastoma cells, and on Drosophila egg laying and development

Salicylhydroxamic acid (SHAM) inhibits the proliferation of cultured plant (Atriplex halimus) and murine neuroblastoma cells with IC50 of 90 and 250 microM, respectively. After 2 h of application, SHAM induces an acceleration of the neuroblastoma cell cycle from G1/S to G2 phases and, after 6 h, it induces an accumulation of the cells in S phase and a cell swelling. Up to 300 microM, SHAM is not cytotoxic and does not induce electrophysiological differentiation of neuroblastoma cells. When Drosophila females are grown in media containing 0.6-1.25 mM SHAM, the rate and number of laid eggs are increased. Furthermore, SHAM stimulates the different development stages from embryo to adult. A general interpretation of the effects of SHAM on cell proliferation and differentiation is proposed.     

Metabolism of biogenic amines in neuroblastoma and glioma cells in culture

The kinetic parameters of monoamine oxidase (MAO; E.C 1.4.3.4) and catechol-O-methyltransferase (COMT; EC 2.1.1.6) were evaluated in extracts of adrenergic and non-adrenergic mouse neuroblastoma cells and in rat glioma cells. Using the naturally-occurring substrates tyramine, tryptamine, serotonin and norepinephrine, the affinity of MAO for a given substrate was independent of the presence of the catecholaminergic pathway or cell type used, with apparent Km values ranging from 8-14 microM for tryptamine to 510-580 microM for norepinephrine. The MAO activity in glioma cells was substantially greater than in either neuroblastoma clone, but Vmax values varied little with substrate among cell lines. Both the neuronal and glial COMT had a similar Km for 1-norepinephrine (200 microM); the corresponding Vmax values were also similar among the different cell lines, but represented only 2-10% of the maximal MAO activity. Neuroblastoma and glioma cells, when grown from early logarithmic to stationary phase, showed no significant changes in specific activity of either MAO or COMT. Growth of cells for 3 days with 1 mM-N6,O2'-dibutyryl adenosine-3',5'-cyclic monophosphate resulted in no marked change in either MAO or COMT activity. These results suggest that in neurons neither MAO nor COMT plays a major role in the type of transmitter inactivation that is analogous to that of acetylcholinesterase in cholinergic synapses. The occurrence of considerable MAO and acetylcholinesterase activities in glioma cells may indicate a role for these cells in neurotransmitter inactivation.     

Turnover of succinyl-CoA:3-oxoacid CoA-transferase in glioma and neuroblastoma cells. Specific influence of acetoacetate in neuroblastoma cells.

The specific activity of succinyl-CoA:3-oxo-acid CoA-transferase (3-oxoacid CoA-transferase, EC 2.8.3.5) increases significantly during growth in culture in both mouse neuroblastoma N2a and rat glioma C6 cells. To investigate the mechanism(s) responsible for this, antibody specific for rat brain 3-oxoacid CoA-transferase was raised in rabbits. Immunotitrations of 3-oxoacid CoA-transferase from neuroblastoma and glioma cells on days 3 and 7 of growth after subculture showed that the ratio of 3-oxoacid CoA-transferase activity to immunoprecipitable enzyme protein remained constant, indicating that differences in specific activity of the enzyme at these times in both cell types reflect differences in concentration of enzyme protein. In glioma cells, the relative rate of 3-oxoacid CoA-transferase synthesis was about 0.04-0.05% throughout 9 days in culture. In contrast, the relative rate of synthesis of 3-oxo-acid CoA-transferase in neuroblastoma cells was about 0.07-0.08% on days 3, 5 and 7 after subculture, but fell to 0.052% on day 9. The degradation rates of total cellular protein (t1/2 = 28 h) and 3-oxoacid CoA-transferase (t1/2 = 46-50 h) were similar in both cell lines. The rise in specific activity of the enzyme in both cell lines from days 3 to 7 without a significant increase in the relative rate of synthesis reflects a slow approach to steady-state conditions for the enzyme secondary to its slow degradation. Differences in 3-oxoacid CoA-transferase specific activity between the two cell lines are apparently due to a difference of about 60% in relative rates of enzyme synthesis. The presence of 0.5 mM-acetoacetate in the medium significantly increased the specific activity of 3-oxoacid CoA-transferase in neuroblastoma cells during the early exponential growth phase. This treatment increased the relative rate of synthesis of 3-oxoacid CoA-transferase by 23% (P less than 0.025) in these cells on day 3, suggesting that substrate-mediated induction of enzyme synthesis is a mechanism of regulation of 3-oxoacid CoA-transferase.     

Regulation by retinoic acid of ICAM-1 expression on human tumor cell lines.

In a group of four human tumor cell lines comprising one melanoma, one glioma, one teratocarcinoma and one neuroblastoma, the expression of the intercellular adhesion molecule-1 (ICAM-1) was found to be significantly increased following treatment with 10 microM of all-trans retinoic acid. In the melanoma and glioma cell lines HS 294T and HS 683, greater than 90% of the cells reacted with the anti-ICAM-1 monoclonal antibody (mAb) CL203.4 in the absence of treatment. Retinoic acid increased the cell surface expression of the molecule by 2-fold. In the teratocarcinoma and neuroblastoma cell lines, TERA-2 and SK-N-SH, the constitutive expression of ICAM-1 was weak, the percentage of cells stained above the background being less than 25%. Retinoic acid induced ICAM-1 expression in greater than 80% of the cells and increased the levels of expression by 2.5 to 3-fold. Immunoprecipitation studies in biosynthetically labeled cells as well as RNase protection analysis confirmed that retinoic acid treatment increased the amount of ICAM-1 at both the protein and mRNA level. The induction or stimulation occurred within 24 h, was maximal after 4 days and reversible.     

Induction of apoptosis in rat C6 glioma cells by panaxydol

Panaxydol is a naturally occurring non-peptidyl small molecule isolated from the lipophilic fractions of Panax notoginseng, a well-known Chinese traditional medicine. Previous studies have shown that panaxydol inhibited the growth of various kinds of malignant cell lines. To date, there has been no report concerning the effect of panaxydol on cell growth inhibition in glioma cells. In this paper, we examined panaxydol's antiproliferation and proapoptotic effects on rat C6 glioma cells and investigated its mechanism. Cell growth inhibition of panaxydol was determined by MTT reduction assay. Apoptosis of cells was measured by both Hoechst 33258 staining and Annexin V analysis. It was found that panaxydol markedly inhibited proliferation of C6 cells in a dose-dependent manner with ID(50) of 40 microM. The cell apoptosis was observed at 48 h in the presence of panaxydol. In concert with these findings, Western blot analysis showed a decreased expression of bcl-2 and increased levels of Bax and caspase-3 in C6 cells treated by panaxydol. In conclusion, panaxydol has profound effects on growth and apoptosis of C6 cells, suggesting that panaxydol may be a potential candidate for the treatment of malignant gliomas.     

Substance P enhances cation permeability of neuronal cell lines

Substance P stimulated the uptake of guanidinium in neuroblastoma X glioma hybrid cells and neuroblastoma cells but not in polyploid glioma cells. Guanidinium has previously been shown to pass the action potential Na+ channel in the two neuronal cell lines. Half-maximal stimulation was reached at 3 microM substance P and, with the hybrid cells, a saturation was seen above 10 microM. The analogue (D-Pro2,D-Trp7,9)-substance P, recently described as a substance P antagonist, caused a stimulation of guanidinium uptake comparable to that seen in the presence of substance P and did not inhibit the stimulation exerted by substance P. The pharmacological properties of the substance P-activated ion channel were investigated. Tubocurarine, phentolamine and propranolol blocked the substance P-stimulated guanidinium uptake with half-maximal inhibitory concentrations of 0.5, 5 and 50 microM. A similar characteristics has been found previously with the veratridine-activated Na+ channel in the cell lines investigated here. Peptides structurally related to substance P such as physalaemin and eledoisin, or others such as neurotensin, bradykinin, D-Ala2, Met5-enkephalinamide and ACTH(1-24) did not affect guanidinium uptake. In view of the high concentrations of substance P required for eliciting an effect in the cell lines, the involvement of specific receptors is questioned. A direct interaction of the peptide with the action potential Na+ channel is discussed.     

Neuroblasts-glia interaction. The effect of co-cultivation upon ecto-ATPase activity of neuroblastoma and glioma cells.

The effect of cell contact and cell medium upon the ecto-enzymes, Mg²⁺- and Ca²⁺-dependent ATPase and 5′-nucleotidase were studied in nervous system cells in tissue culture. Conditions were worked out for co-culture and rseparation of glioblastoma and neuroblastoma cells so that the effects upon each of the co-cultured cell lines after interaction of these cells could be reliably determined. Co-cultivation of mouse neuroblastoma and glioma cell lines markedly enhanced Mg²⁺- and Ca²⁺-dependent ecto-ATPase activity. Evidence was obtained which indicates that increase in ecto-ATPase of co-cultured neuro- and glioblastoma cells occurs in both cell types. Ecto-ATPase was 500% of the original level in clonal line NN astroblasts after co-culture with M1 neuroblasts. This activity decreased over 50 transfers during the period of about a year. Increase in ecto-ATPase and morphological differentiation of M1 neuroblastoma cells after co-culture with NN astroblasts could also be brought about simply by treatment with the medium from NN cell cultures. Co-cultivation of neuroblastoma and glioma cells does not change significantly the specific activity of ecto-5′-nucleotidase.     

Increased sensitivity to the platelet-derived growth factor (PDGF) receptor inhibitor STI571 in chemoresistant glioma cells is associated with enhanced PDGF-BB-mediated signaling and STI571-induced Akt inactivation

The platelet-derived growth factor receptor (PDGFR) is a tyrosine kinase, implicated in the development and progression of different tumors, including gliomas. Chemoresistance is a common feature of malignant gliomas. Since receptor tyrosine kinases contribute to chemoresistance in tumors, we addressed whether PDGFR signaling might confer selective growth advantage to chemoresistant cells. The effects of the PDGFR inhibitor STI571 on proliferation and PDGFR signaling were compared in chemosensitive and cisplatin-selected, chemoresistant sublines derived from glioma and from two other PDGFR-expressing tumors (ovarian carcinoma and neuroblastoma). The chemoresistant glioma U87/Pt cells were twofold more sensitive to STI571 growth-inhibitory effects than the chemosensitive U87 cells, and two- to threefold more sensitive than five unrelated glioma cell lines. The other two paired cell lines were equally responsive. Sensitization of U87/Pt cells correlated with upregulation of the PDGF-B isoform and with PDGF-BB-induced Akt overactivation, which was prevented by STI571. STI571 specifically inhibited PDGF-BB-, but not PDGF-AA- or stem cell factor-mediated signaling. In serum-containing medium, STI571 decreased phospho-Akt in U87/Pt cells, but not in U87, while activating extracellular signal-regulated kinase (Erk) in both. STI571 antiproliferative effects were partially reverted by constitutively active Akt. Cotreatment with inhibitors of phosphatidylinositol 3'-kinase (PI3K) or mitogen-activated protein kinase kinase (MEK) resulted in enhanced growth inhibition in glioma cells. Our results suggest that increased PDGF-BB signaling may sensitize chemoresistant glioma cells to STI571, suggesting a therapeutic potential for STI571 in patients with malignant gliomas refractory to chemotherapy. Simultaneous blockade of PDGFR and PI3K or Erk pathway may enhance therapeutic targeting in gliomas.     

Effects of retinoic acid (RA) on the growth and phenotypic expression of several human neuroblastoma cell lines

It has been shown that retinoic acid (RA) can promote morphologic differentiation and inhibit the growth of a human neuroblastoma cell line, LA-N-1. The present study tests the histological generality of these phenomena by determining the effects of RA on seven other human neuroblastoma cell lines. Results show that RA strongly inhibited anchorage-dependent growth and induced morphologic alterations in six of seven of the cell lines. These alterations included morphologic differentiation as evidenced by formation of neurite extensions in four of the lines, cellular enlargement and vacuolization in one culture, and formation of large, flattened epithelial or fibroblastic-like cells in another culture. Although one cell line was relatively insensitive to the effects of RA in monolayer culture, all seven were strongly inhibited by RA in soft agar assays. Cellular RA-binding proteins were detected in 2/2 lines tested. These findings suggest that, as a histological group, human neuroblastoma cells are extremely sensitive to RA-induced growth inhibition and morphological alterations generally associated with reduced expression of the malignant phenotype of this type of cancer.     

Glycolytic metabolism in cultured cells of the nervous system

The effects of thiamine deficiency and of the antithiamine drug pyrithiamine on the C-6 glioma and the C-1300 neuroblastoma cell lines have been studied. Thiamine deficiency increased the doubling time of the neuroblastoma cells without affecting that of the glioma cells. Pyrithiamine prevented both cell lines from doubling even once. (hiamine deficiency had only slight effects on intracellular pyruvate and lactate levels or on efflux rates for the acids, but pyrithiamine treatment resulted in large increases in both the intracellular levels and the efflux in both cell lines. For comparison, the pyruvate and lactate levels in mouse brain were measured. The levels from thiamine-deficient mouse brain were essentially unchanged from controls while pyrithiamine treatment caused a significant elevation only of the pyruvate concentration.     

Density-dependent inhibition of 2-deoxy-d-glucose uptake into glioma and neuroblastoma cells in culture

The transport of 2-deoxy-d-glucose (2-DG) into cultured human glioma (138 MG) and mouse neuroblastoma (C1300) cells has been studied in relation to the growth curves of the cells. An initial increase in the uptake of 2-DG into exponentially growing 138 MG cells, could be attributed to the number of days the cells were kept in culture rather than to their increased density. As the 138 MG cells reached confluency after 3 days the 2-DG uptake became density-dependent inhibited. In still denser cultures the cell growth was inhibited. This was accompanied by morphological ‘normalization’ of the cells and increased uptake of 2-DG. Uptake of 2-DG into C1300 cells was density-dependent inhibited throughout the cell growth cycle. As the cell density increased from 15 × 10³ to 130× 10³ cells/cm² the rate of uptake/cell decreased to one-fourth. At the latter cell density the cells entered stationary phase, without any major changes in morphology. The results suggest that spontaneously occurring tumour cells, such as glioma and neuroblastoma cells, can regulate the sugar transport in relation to cell density. This could be due to newly-acquired differentiated properties of the cells or to true contact-inhibitory phenomena.     

Comparison of growth inhibition profiles and mechanisms of apoptosis induction in human colon cancer cell lines by isothiocyanates and indoles from Brassicaceae

The isothiocyanates sulforaphane and PEITC (beta-phenethyl isothiocyanate) as well as the indoles indole-3-carbinol and its condensation product 3,3'-diindolylmethane are known to inhibit cancer cell proliferation and induce apoptosis. In this study, we compared the cell growth inhibitory potential of the four compounds on the p53 wild type human colon cancer cell line 40-16 (p53(+/+)) and its p53 knockout derivative 379.2 (p53(-/-)) (both derived from HCT116). Using sulforhodamin B staining to assess cell proliferation, we found that the isothiocyanates were strongly cytotoxic, whereas the indoles inhibited cell growth in a cytostatic manner. Half-maximal inhibitory concentrations of all four compounds in both cell lines ranged from 5-15 microM after 24, 48 and 72 h of treatment. Apoptosis induction was analyzed by immunoblotting of poly(ADP-ribose)polymerase (PARP). Treatment with sulforaphane (15 microM), PEITC (10 microM), indole-3-carbinol (10 microM) and 3,3'-diindolylmethane (10 microM) induced PARP cleavage after 24 and 48 h in both 40-16 and the 379.2 cell lines, suggestive of a p53-independent mechanism of apoptosis induction. In cultured 40-16 cells, activation of caspase-9 and -7 detected by Western blotting indicated involvement of the mitochondrial pathway. We detected time- and concentration-dependent changes in protein expression of anti-apoptotic Bcl-x(L) as well as pro-apoptotic Bax and Bak proteins. Of note is that for sulforaphane only, ratios of pro- to anti-apoptotic Bcl-2 family protein levels directly correlated with apoptosis induction measured by PARP cleavage. Taken together, we demonstrated that the glucosinolate breakdown products investigated in this study have distinct profiles of cell growth inhibition, potential to induce p53-independent apoptosis and to modulate Bcl-2 family protein expression in human colon cancer cell lines.     

Differentiation of neuroblastoma cells induced by an inhibitor of mevalonate synthesis: relation of neurite outgrowth and acetylcholinesterase activity to changes in cell proliferation and blocked isoprenoid synthesis

Mevinolin, a competitive inhibitor of 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase, stimulates neurite outgrowth and acetylcholinesterase (ACE) activity in C1300 (Neuro-2A) murine neuroblastoma cells. Sprouting of neurites began within 4-8 h, before changes in cell proliferation could be detected by [3H]thymidine incorporation or flow cytometry. In contrast, the increase in ACE activity was temporally correlated with suppression of DNA synthesis, which occurred after 8 h. The activity of the membrane marker enzyme phosphodiesterase I was not stimulated by mevinolin. Suppression of protein synthesis with cycloheximide blocked the induction of ACE activity but only partially inhibited neurite outgrowth in the mevinolin-treated cultures. When mevinolin was removed from the culture medium, most of the cells retracted their neurites within 2 h, but ACE activity did not decline until DNA synthesis began to return to control levels after 10 h. Similarly, retraction of neurites in differentiated cells exposed to colchicine was not accompanied by a decrease in ACE activity. DNA histograms suggested that mevinolin arrests neuroblastoma cells in both the G1 and G2/M compartments of the cell cycle. Other cytostatic drugs that arrest cells at different stages of the cell cycle did not cause Neuro-2A cells to form neurites such as those seen in the mevinolin-treated cultures. When incorporation of [3H]acetate into isoprenoid compounds was studied in cultures containing mevinolin in concentrations ranging from 0.25 microM to 25 microM, the labeling of cholesterol, dolichol, and ubiquinone was suppressed by 90% or more at all concentrations. However, significant growth arrest and cell differentiation were observed only at the highest concentrations of mevinolin. Supplementing the medium with 100 microM mevalonate prevented the cellular response to mevinolin, but additions of cholesterol, dolichol, ubiquinone, or isopentenyl adenine were generally ineffective. The cholesterol content of neuroblastoma cells incubated with 25 microM mevinolin for 24 h was not diminished, and protein glycosylation, measured by [3H]mannose incorporation, was decreased only after 24 h at high mevinolin concentration. These studies suggest that the stimulation of neurite outgrowth and the increase in ACE activity induced by mevinolin are independent phenomena. Whereas neurite outgrowth is not related directly to the effects of mevinolin on cell cycling, the induction of ACE is correlated with the inhibition of cell proliferation.(ABSTRACT TRUNCATED AT 400 WORDS)     

Cytotoxicity of organophosphate anticholinesterases

Summary Organophosphate (OP) anticholinesterases were found to modulate metabolic activities of human neuroblastoma cells and hepatocytes, which was detectable by the Cytosensor? microphysiometer. The nerve gas ethyl-S-2-diisopropylaminoethyl methylphosphorothiolate (VX), at 10 μM, produced significant reduction in cell metabolism within 2 min, as measured by changes in the acidification rate of the medium. The reduction was dose-and time-dependent and irreversible after 4 h of exposure. Two alkaline degradation products of VX produced no cytotoxicity. Exposure for 24 h to 3 μM VX caused 36% and 94% irreversible loss of metabolism in hepatocytes and neuroblastoma cells, respectively. The insecticides parathion and chlorpyrifos stimulated hepatocyte metabolism but inhibited neuroblastoma cells. Their oxons were more active. Exposure of neuroblastoma cells for 4 h to VX, parathion, paraoxon, diisopropylfluorophosphate or chlorpyrifos gave an LC₅₀ of 65, 775, 640, 340, or 672 μM, respectively, whereas 24 h gave an LC₅₀ of 0.7, 3.7, 2.5, 29, and 31 μM, respectively. Preincubation of hepatocytes with phenobarbital enhanced their response to parathion and VX due to metabolic bioactivation. Atropine partially blocked the effects of VX and paraoxon on both cell types, which suggests the involvement of a muscarinic receptor as the target for cytotoxicity. There was no correlation between OP in vivo neurotoxicity and in vitro cytotoxicity. It is suggested that the former results from their cholinesterase inhibition, while the latter results from action on different targets and requires much higher concentrations.     

Forskolin induction of S-100 protein in glioma and hybrid cells

The S-100 protein level in mouse neuroblastoma (N18TG-2 and NIE-115), rat glioma (C6, C6BU-1, and C6V-1), and hybrid (NG108-15, 140-3, 141-B, NBr10A, NBr20A, NCB20, and NX3IT) cells was determined with a sensitive enzyme immunoassay system that uses a rabbit antibody to bovine brain S-100 protein. S-100 protein was detected in glioma but not in neuroblastoma cells. All seven hybrid cells derived from neuroblastoma and glioma or other types of cells were found to possess a very little or undetectable S-100 protein. The induction of S-100 protein level in prestationary phase cultures of glioma C6BU-1 cells was examined by forskolin, which was a highly specific activator of adenylate cyclase of the cells and produced morphological differentiation. After incubation with 10 microM forskolin for 48 hr, the S-100 protein level increased 2-2.5-fold in C6BU-1 glioma cells whose mean control level was 60 +/- 26 ng/mg protein (+/- SD). The forskolin induction of S-100 protein in the cells was dose dependent, and the concentration of forskolin required for 50% activation of S-100 protein was about 0.6 microM. The increase by forskolin was initiated from 10-15 hr after incubation with it and was inhibited with cycloheximide and actinomycin D. In NG108-15 hybrid cells the induction of S-100 protein was also observed by forskolin as well as prostaglandin (PG) E1 plus theophylline which are known to activate adenylate cyclase of the cells. The results indicate that S-100 protein biosynthesis is genetically controlled in these clonal cells, and that S-100 protein can be regulated in a cAMP-dependent fashion in prestationary cultures.     

Differential effects of tumor necrosis factor on the growth and differentiation of neuroblastoma and glioma cells

We have studied the effect of tumor necrosis factor (TNF-alpha) on transformed neural and glial-derived cell lines. TNF-alpha at physiological doses was able to arrest the growth and inhibit DNA synthesis of N103 neuroblastoma cells. This phenomenon was accompanied by a morphological cell differentiation characterized by the outgrowth of neurites. By contrast, TNF-alpha induced an increase in the growth rate of C6 glioma cells and upon cytokine addition a higher number of C6 cells were found in the S + G2 phase of the cell cycle. C6 cells did not show morphological changes under this treatment. Analogous results were obtained with IFN-gamma. These neurotrophic and mitogenic effects of TNF-alpha suggest a putative role of this cytokine in the regeneration of brain tissue upon brain injury.