Development of a monoclonal antibody against a tumor-associated antigen.

A monoclonal antibody-producing hybrid cell line was obtained by fusing mouse myeloma cells with spleen cells from a mouse immunized with C6 glioma cells. This antibody binds to a specific cell-surface antigen that is present on C6 rat glioma cells, transformed astrocytes and oligodendrocytes, and a human glioma cell line but is absent on a normal glial cell line, fibroblasts, and primary cultures of astrocytes and oligodendrocytes. The antigen also appears on tumor tissue of transformed oligodendrocytes but not on normal brain tissue.

Neoplastic transformation of newborn rat oligodendrocytes in culture.

We have developed a model to study the neoplastic transformation of rat oligodendrocytes in culture. This procedure utilizes a technique previously developed by McCarthy and de Vellis which allows the preparation of 99% pure astrocyte and oligodendrocyte populations from 1- to 2-day-old rat cerebral cortices. Pregnant rats on the 19th day of gestation were given injections with either ethyl nitrosourea (10 micrograms/g body weight) in phosphate-buffered saline or phosphate-buffered saline, and oligodendrocyte cultures were prepared. Oligodendrocytes appear to be unstable in culture since transformation was observed with cells derived from either pups from pregnant rats either treated with nitrosourea or phosphate-buffered saline. Transformation required 78 to 108 days and 3 to 9 passages, at which time a marked increase in cellular proliferation was observed. The possibility that the transformed cells were derived from a nonoligodendroglial cell was excluded by the following evidence. Light and scanning electron micrographs of the transformed cells revealed cytological features essentially similar to those of primary oligodendroglial cultures. Furthermore, 2 biochemical oligodendroglial markers, the induction of lactate dehydrogenase by N6,O6-dibutyryl cyclic adenosine 3':5'-monophosphate and the presence of 2',3'-cyclic nucleotide 3' phosphohydrolase, were also retained. Conversely, another oligodendroglial marker, the hydrocortisone induction of glycerol phosphate dehydrogenase, was not found in any of the cell lines. These transformed cells grew as tumors when injected intracranially into 21-day-old rats. Histologically, these tumors did not appear as classical oligodendrogliomas, but their oligodendroglial origin was confirmed since the tumor tissue contained 2':3'-cyclic nucleotide 3'-phosphohydrolase activity, and the cells which grew from tumor explant cultures morphologically appeared similar to the parent cell line. The transformed cells were also characterized for in vitro properties which correlate with the expression of tumorigenicity. The transformed cells exhibited anchorage-independent growth and were agglutinated by concanavalin A treatment. Changes in fibrinolytic activity were not an exclusive property of transformed glial cells. This model should now allow us to study various mechanisms involved in the neoplastic transformation of oligodendrocytes.

Second stage tumor promoters: differences in biological potency and phorbol ester receptor affinity in C6 cells.

We have shown that the second stage tumor promoters mezerein (MEZ) and phorbol 12-retinoate 13-acetate (PRA) inhibit the gluccocorticoid-induced increase in glycerol phosphate dehydrogenase (GPDH) activity in C6 rat glioma cells with ED 50-values of 3.9 and 2.9 nM, respectively. Phorbol 12-myristate 13-acetate (PMA) was 10-fold less potent. MEZ was likewise more potent than PMA for inhibition of cAMP formation in response to isoproterenol. Binding competition studies using [3H]phorbol 12,13-dibutyrate ([3H]PDBu) yielded apparent Ki-values for MEZ and PRA of 50-70 nM. The large difference between the biological potencies of MEZ and PRA and their affinity for the major phorbol ester receptor suggest they may be acting through a more complicated mechanism in these cells.

Mechanisms of lead neurotoxicity.

During the past several years, there has been a renewed interest in the mechanisms by which lead poisoning disrupts brain function. In part, this is related to clinical observations that imply an absence of threshold for toxicity in the immature brain. Many of the neurotoxic effects of lead appear related to the ability of lead to mimic or in some cases inhibit the action of calcium as a regulator of cell function. At a neuronal level, exposure to lead alters the release of neurotransmitter from presynaptic nerve endings. Spontaneous release is enhanced and evoked release is inhibited. The former may be due to activation of protein kinases in the nerve endings and the latter to blockade of voltage-dependent calcium channels. This disruption of neuronal activity may, in turn, alter the developmental processes of synapse formation and result in a less efficient brain with cognitive deficits. Brain homeostatic mechanisms are disrupted by exposure to higher levels of lead. The final pathway appears to be a breakdown in the blood-brain barrier. Again, the ability of lead to mimic or mobilize calcium and activate protein kinases may alter the behavior of endothelial cells in immature brain and disrupt the barrier. In addition to a direct toxic effect upon the endothelial cells, lead may alter indirectly the microvasculature by damaging the astrocytes that provide signals for the maintenance of blood-brain barrier integrity.

Associations of lead exposure and dose measures with erythrocyte protein kinase C activity in 212 current Korean lead workers.

Lead can replace calcium in enzyme assays that measure protein kinase C activity and lead activates protein kinase C in human erythrocytes after exposure to lead in vitro. To examine the relevance of these observations to lead exposure in humans, we studied the associations of lead found in blood or tibia with activation of protein kinase C in erythrocytes isolated from workers in the lead industry. We examined erythrocytes among 212 lead workers, with a mean (+/-SD) age of 39.1 (10.0) years and exposure duration of 8.1 (6.5) years and measured protein kinase C activation by an in vitro back-phosphorylation assay. After adjustment for potential confounding factors (age and sex), tibia lead and exposure duration were significantly associated with erythrocyte protein kinase C activation (both p values < 0.05). No associations were observed between protein kinase C activation and blood-lead or zinc-protoporphyrin levels. These findings suggest that human exposure to lead results in activation of erythrocyte protein kinase C, which may be directly relevant to the neurotoxicity of lead.

Neoplastic transformation of newborn rat astrocytes in culture.

A subpopulation of rat glial cells, derived from the astroglial population of newborn cerebral cortex cell cultures, spontaneously transformed in culture. Unlike the pretransformed cells, the transformed cells formed pile-up colonies, exhibited anchorage-independent growth, and were tumorigenic in young rats. Both the pretransformed and transformed cells exhibited differentiated properties characteristic of glial cells. For example, the pretransformed cells possessed hydrocortisone-inducible glutamine synthetase (GS), a property restricted to astrocytes in the central nervous system. As was anticipated, these cells did not exhibit either of two oligodendroglial characteristics, hydrocortisone-inducible glycerol phosphate dehydrogenase (GPDH) or the induction of lactate dehydrogenase (LDH) by N6,O6-dibutyryl cyclic adenosine 3':5'monophosphate (Bt2 cAMP). Unexpectedly, the transformed cells expressed the induction of glycerol phosphate dehydrogenase and lactate dehydrogenase but lost the glutamine synthetase induction. Both the pretransformed and transformed cells were examined ultrastructurally. Neither cell type exhibited glial filaments (9-10 nm), a structure typical of astrocytes. Rather, the pretransformed cells were characterized by distinct longitudinal filaments near the cell surface and the absence of microtubules. On the other hand, the only cytoskeletal element visible in transformed cells were microtubules. Our work demonstrates that, like other rodent cell types, rat glial cells can spontaneously transform in culture. It also shows that the expression of differentiated properties are sensitive to the transformation process.

Chemotaxis of rat brain astrocytes to platelet derived growth factor.

Using a purified population of rat brain astrocytes prepared from neonatal cortex, we investigated the chemotaxis of astroglia to several well characterized growth factors. Chemotactic activity for astrocytes was found for the platelet derived growth factor with a half maximal response occurring at 0.5 ng/ml as compared with a value of 2-3 ng/ml obtained for NIH/3T3 fibroblasts in control experiments. Other growth factors including epidermal growth factor, fibroblast growth factor and insulin were inactive as chemoattractants. Affinity purified fibronectin was also found to stimulate the migration of astroglia, with half maximal doses of approximately 1 microgram/ml relative to maximal responses to platelet derived growth factor.

Induction of c-fos mRNA by lead in PC 12 cells.

Addition of lead acetate to PC 12 pheochromocytoma cells elicits induction of c-fos, an immediate early response gene. Induction of c-fos was concentration- and time-dependent: the lowest concentration of lead acetate tested that induced c-fos was 10 microM; induction was observed after a 30 min incubation and remained high after 90 min. Treatment with lead acetate and cycloheximide superinduced c-fos mRNA. Actinomycin D, an inhibitor of mRNA transcription, decreased the level of c-fos mRNA induced by lead acetate by almost 80%. Cadmium chloride and zinc chloride did not induce c-fos mRNA. Since the c-fos gene encodes a transcription factor, Pb2+ has the potential to deregulate the expression of other genes.

Effect of ganglioside GM1 on arachidonic acid release in bovine aortic endothelial cells.

A role for the ganglioside GM1 in arachidonic acid release in bovine aortic endothelial cells (BAEC) was investigated. [3H]Arachidonic acid-labeled BAEC were preincubated with GM1 and incubated with one of four different stimulators. GM1 inhibited arachidonic acid release when stimulated with maitotoxin or melittin but not with ionomycin or thapsigargin. A 10 microM GM1 concentration achieved a 50% and 100% inhibition of the maitotoxin and melittin responses, respectively. The selective inhibition displayed by GM1 on the maitotoxin and melittin responses was not due to its ability to bind calcium since all four drugs, maitotoxin, melittin, ionomycin, and thapsigargin, required extracellular calcium. The effect of GM1 was not specific to arachidonic acid release. GM1 at 50 microM inhibited phosphatidylinositol polyphosphate (PIP) hydrolysis mediated by melittin, but did not affect hydrolysis mediated by ionomycin. Moreover, the activity of GM1 was not restricted to phospholipid metabolism since it also inhibited calcium influx that was stimulated by maitotoxin or melittin but not by ionomycin. We conclude that GM1 is not a specific inhibitor of phospholipases in bovine aortic endothelial cells, but rather its activity is dependent on the type of stimulant used to activate the cell.

Retinal microvessels express less gamma-glutamyl transpeptidase than brain microvessels.

In this investigation we localized and compared the level of gamma-glutamyl transpeptidase (GGTP) activity in retinal and brain preparations using histochemical, enzymatic and in situ hybridization assays. We compared GGTP distribution to another microvessel specific enzyme, alkaline phosphatase (AP). In the rat brain, GGTP activity was observed in microvessels and choroid plexus by a histochemical method. Similar studies in the rat retina revealed activity in the pigment epithelium but only a very weak reaction in microvessels. Histochemical staining for alkaline phosphatase was observed in both retinal and brain microvessels choroid plexus and pigment epithelium. Biochemical analysis verified that GGTP activity was significantly lower in retinal than brain microvessels, while alkaline phosphatase activity was similar in both types of microvessels. GGTP specific activity of bovine brain and retinal microvessels was 185 +/- 39 mUnits and 8.5 +/- 1.5 mUnits (p less than 0.001), respectively. By contrast, alkaline phosphatase specific activity in brain and retinal microvessels was 732 +/- 139 and 471 +/- 114 (p greater than 0.1), respectively. Choroid plexus and retinal pigment epithelium exhibited similar levels of GGTP and alkaline phosphatase. Differences in GGTP expression between retinal and brain microvessels were also observed on the mRNA level. In situ hybridization studies revealed that brain microvessels expressed four times more GGTP specific mRNA than retinal microvessels. We conclude that retinal microvessels do not express high levels of GGTP which may make them more vulnerable than brain microvessels to injuries mediated by leukotrienes and oxidative stress.

Regulation of cAMP levels by protein kinase C in C6 rat glioma cells.

Cultures of rat C6 rat glioma cells exhibit a diminished response to isoproterenol and forskolin after being treated with phorbol 12,13-dibutyrate (PDbU). An IC50 for PDbU of 38 +/- 5 nM and 62 +/- 8 nM was observed in the isoproterenol and forskolin response, respectively. Similarly, C6 cultures exhibited a diminished response to isoproterenol and forskolin after an overnight incubation with phospholipase C. We previously demonstrated that this treatment will increase diacylglycerol levels in these cells (Bressler: J Neurochem 48:181-186, 1987). An IC50 for phospholipase C of 6.0 +/- 0.1 x 10(-1) and 7.0 +/- 0.1 x 10(-1) units/ml was observed for the isoproterenol and forskolin response, respectively. A kinetic analysis suggests that the site of PDbU-mediated inhibition to beta-adrenergic and forskolin stimulation was different. Degradation of cAMP was a contributory factor since elevated cAMP levels decreased faster in PDbU treated cells than in nontreated cells. In addition, PDbU treated cells exhibited a significantly higher level of phosphodiesterase activity. We conclude that activation of protein kinase C and subsequent stimulation of phosphodiesterase activity contributes to the inhibition of the beta-adrenergic and forskolin mediated increase in cAMP levels in intact C6 rat glioma cells. The consequences of lower cAMP levels in sustaining differentiated function in the C6 rat glioma cell line will be discussed.

Glial shape and cytoskeletal protein synthesis.

We investigated whether the shape of astroglial derived cells influences the expression of cytoskeletal proteins. In reaggregating cultures GFAP, vimentin and actin synthesis was approximately 52%, 50% and 37% the level found in monolayer cultures, respectively. Monolayer cultures consisted of polygonal shaped cells adhering to plastic, while reaggregating cultures were comprised of round cells growing in a suspension like culture. Additionally, human glioma cells induced to grow as round cells on poly-2-hydroxyethyl methacrylate (polyhema) coated plastic exhibited a level of GFAP synthesis that was approximately 20% the level displayed by polygonal shaped cells grown on uncoated plastic. Glioma cells initially grown on a polyhema surface and replated onto uncoated plastic were capable of reinitiating GFAP synthesis. Thus, alterations in the synthesis of GFAP and other cytoskeletal proteins can occur when astrocytes change their shape.

Role of anion exchange and thiol groups in the regulation of potassium efflux by lead in human erythrocytes.

Pb2+ is thought to enter erythrocytes through anion exchange (AE) and to remain in the cell by binding to thiol groups. To define the role of AE mechanisms and thiol groups in Pb2+ toxicity, we studied the effects of drugs and conditions that modify AE and that modify thiol groups on the ability of Pb2+ to stimulate potassium efflux as measured with 86Rb. The most potent stimulation of 86Rb efflux by Pb2+ occurred when conditions were optimal for the AE mechanism--that is, when bicarbonate was included in the buffer or a buffer made with Nal or NaCl rather than NaClO4 or NaNO3 was used. Furthermore, 4,4'-diisothiocyanatostilbene-2,2'-disulfonic acid and 4-acetamido-4'-isothiocyanatostilbene-2,2'-disulfuonic acid, potent inhibitors of the AE mechanism, completely inhibited stimulation of the 86Rb efflux by Pb2+. These conditions or inhibitors did not affect stimulation of the 86Rb efflux by ionomycin plus Ca2+. A role for Ca2+ channels was dismissed because the inorganic Ca2+ channel blockers, Cd2+ or Mn2+, did not prevent stimulation of 86Rb efflux by Pb2+ but did inhibit stimulation by ionomycin plus Ca2+. 86Rb efflux was more sensitive to Pb2+ if erythrocytes were treated for 15 min with thiol-modifying reagents that enter cells, such as iodoacetamide, N-ethylmaleimide, or dithiothreitol, than to reduced glutathione, a thiol-modifying reagent that is not permeable to the cell. Thus, in erythrocytes the AE mechanism and internal thiol groups are critical factors that affect the stimulation of a Ca(2+)-dependent process by Pb2+.

Glucocorticoid-mediated increases in glycerol phosphate dehydrogenase activity is inhibited by the phorbol ester tumor promoters.

An oligodendroglial specific property, glucocorticoid regulation of glycerol-3-phosphate dehydrogenase (GPDH) levels, was inhibited in C6 rat glioma cells when 4 beta-phorbol 12-myristate 13-acetate (PMA) was added to the cultures. PMA inhibited GPDH induction in both logarithmic- and stationary-phase cells. These events are most likely mediated through the phorbol ester receptor since the ability of various phorbol ester analogs to compete with the ligand [3H]4 beta-phorbol 12,13-dibutyrate for binding to the receptor correlates with the ability the particular analog has to inhibit GPDH induction. Additionally, like tumor promotion in vivo, the inhibition of GPDH induction is reversible. The PMA effect is not restricted to the C6 cell line since PMA also inhibits GPDH inducibility in another rat glioma cell line. This PMA-mediated event has been partially characterized. PMA did not affect the overall rate of protein or RNA synthesis. It was ineffective in altering both glucocorticoid accumulation to the nucleus and the rate of GPDH degradation. It appears likely that PMA's inhibitory action occurs at the transcriptional or translational level.

Sex-related immunocompetence of BALB/c mice. I. Study of immunologic responsiveness of neonatal, weanling, and young adult mice.

The responses of lymphoid cells from the thymus, lymph nodes, and spleen of male and female BALB/c mice were evaluated to determine if sex-related variations in immune expression could be found. Immunologic assays used included blastogenic responses to mitogens, mixed lymphocyte responses, and direct and indirect measurement of plaque-forming cells against soluble and particulate antigens. The results indicated that responses of spleen cells from young adult female mice were higher than those of males in all comparative tests. Little or no differences between the sexes were observed in the mitogenesis of lymph nodes and thymuses. Newborn mice did not demonstrate the sex-associated immune differences. Among the weanling mice slight differences between male and female spleen cells responsiveness to mitogenic agents were observed.

Inhibition of astroglia-induced endothelial differentiation by inorganic lead: a role for protein kinase C.

Microvascular endothelial function in developing brain is particularly sensitive to lead toxicity, and it has been hypothesized that this results from the modulation of protein kinase C (PKC) by lead. We examined the effects of inorganic lead on an in vitro model of central nervous system endothelial differentiation in which astroglial cells induce central nervous system endothelial cells to form capillary-like structures. Capillary-like structure formation within C6 astroglial-endothelial cocultures was inhibited by lead acetate with 50% maximal inhibition at 0.5 microM total lead. Inhibition was independent of effects on cell viability or growth. Under conditions that inhibited capillary-like structure formation, we found that lead increased membrane-associated PKC in both C6 astroglial and endothelial cells. Prolonged exposure of C6 cells to 5 microM lead for up to 16 h resulted in a time-dependent increase in membranous PKC as determined by immunoblot analysis. Membranous PKC increased after 5-h exposures to as little as 50 nM lead and was maximal at approximately 1 microM. Phorbol esters were used to determine whether PKC modulation was causally related to the inhibition of endothelial differentiation by lead. Phorbol 12-myristate 13-acetate (10 nM) inhibited capillary-like structure formation by 65 +/- 5%, whereas 4 alpha-phorbol 12,13-didecanoate was without effect. These findings suggest that inorganic lead induces cerebral microvessel dysfunction by interfering with PKC modulation in microvascular endothelial or perivascular astroglial cells.

Gamma-glutamyl transpeptidase activity in brain microvessels exhibits regional heterogeneity.

Brain microvessels form a tight blood-tissue permeability barrier and express high levels of specific enzymes, including gamma-glutamyl transpeptidase (GGTP). This differentiation is thought to be induced by perivascular astrocytes. By using histochemical methods, we found that the percentage of GGTP-positive vessels varied considerably in different areas of rat brain. Enzyme activity was not found in the pineal gland or the median eminence, where the blood-brain barrier is not expressed. In areas where the blood-brain barrier is expressed, the percentage of GGTP-positive vessels varied from 8% in the optic nerve to 100% in the anterior commissure. The neocortex showed a lower percentage of GGTP-positive vessels (2-15%) than anterior olfactory nucleus (42%), subiculum (70%), hippocampus (48%), and striatum (50-58%). Alkaline phosphatase, another brain microvessel-enriched enzyme, did not show these marked regional differences. The morphometric histochemical results were verified by enzymatic assays in homogenates of different regions from rat and bovine brain and in microvessel preparations of bovine putamen and neocortex. During the postnatal development of rat brain, the difference between neocortex and striatum appeared after day 20. The regional heterogeneity of brain microvessels may be caused by astrocytic heterogeneity and reflect regional heterogeneity in microvascular function.

Astroglial membrane structure is affected by agents that raise cyclic AMP and by phosphatidylcholine phospholipase C.

The role of signal transduction mechanisms in the production of the characteristic orthogonal arrays of particle assemblies in the astroglial plasma membrane was investigated in vitro by freeze-fracture electron microscopy. Agents which raise cellular cAMP levels and subsequently activate protein kinase A, such as forskolin (50 microM), isoproterenol (10 microM) and 8-bromo-cAMP (1 mM), increased the density, the number of assemblies per unit area of cleaved cell membrane, and the frequency of astrocytes with assemblies. Agents that lead to the activation of protein kinase C, such as phorbol 12,13-myristate acetate (at 50 nM) and choline-dependent phospholipase C (at 0.01-0.1 U ml-1), did not affect the assembly concentration. Thus, protein kinase A but not protein kinase C appears to be involved in the production of assemblies or their insertion into the astroglial plasma membrane. Although choline-dependent phospholipase C did not affect the astroglial assemblies, it caused the non-assembly, background particles to aggregate. A choline-dependent phospholipase C from a different source (B. cereus) was also active though at a higher concentration. Phospholipases of different specificities, such as phospholipase A2, phospholipase D or inositol-dependent phospholipase C were inactive over a wide range of concentrations. Two other astroglia derived cells, Müller cells and cells of the C6 glioma cell line, were also similarly affected by choline-dependent phospholipase C, while six other cells types including neurons, endothelial cells and fibroblasts were unaffected. It appears that phosphatidylcholine plays a significant role in determining the membrane structure of astrocytes. In a search for a means of isolating the assemblies, the binding of three lectins: ConA, WGA and PNA, conjugated to gold, was tested by label-fracture to ascertain whether the assemblies have an external oligosaccharide component. None of the lectins bound specifically to assemblies.

Distinct mechanisms of neurotransmitter release from PC 12 cells exposed to lead.

Two enzymes, protein kinase C and microsomal Ca(2+)-ATPase help regulate levels of Ca2+ in many types of cells. Since proteins that regulate Ca2+ often influence sensitivity to Pb2+, we determined the possible roles played by protein kinase C and microsomal Ca(2+)-ATPase for the Pb(2+)-evoked release of norepinephrine (NOR) in PC cells. NOR release was observed at 10 microM Pb2+ when PC 12 cells were stimulated with inhibitors of microsomal Ca(2+)-ATPase such as thapsigargin, cyclopiazonic acid, or 2,5-di-(t-butyl)-hydroquinone. At 5 microM, Pb2+ evoked the release of NOR in PC 12 cells stimulated with activators of protein kinase C such as phorbol 12-myristate 13-acetate (PMA) or (-)-7-octylindolactam. NOR release was observed at 1 microM Pb2+ in the presence of both PMA and thapsigargin. Ni2+ and Cd2+ blocked NOR release stimulated by Pb2+ in the presence of thapsigargin but not by PMA. NOR released by thapsigargin stimulation was not altered in PC 12 cells depleted of protein kinase C. Two proteins found in vesicles, chromogranin B and secretogranin-II were released with NOR. Our results indicate that in PC 12 cells, PB(2+)-evokes the release of neurotransmitters. Furthermore, thapsigargin and PMA increase the cell's sensitivity to Pb2+ by different pathways.

Immediate early gene expression in PC12 cells exposed to lead: requirement for protein kinase C.

We previously demonstrated induction of c-fos mRNA in PC12 cells exposed to lead that was dependent on new transcription. In the current work, we examined two signal transduction mechanisms that are activated by lead and have been shown to mediate induction of c-fos mRNA. One mechanism involves protein kinase C, and the other requires calmodulin-dependent protein kinase II. Significant increases in the levels of c-fos, c-jun, and egr-1 but not NGFIB mRNA were observed in PC12 cells exposed to lead or phorbol 12-myristate 13-acetate. In contrast, PC12 cells depolarized with 56 mM K+ displayed an increase in c-fos, egr-1, and NGFIB but not c-jun mRNA. Similar to other activators of protein kinase C, lead increased AP-1 and Egr-1 DNA binding activity. Additionally, lead increased luciferase activity in cerebellar granule cells transfected with an AP-1 luciferase reporter construct. Lead did not increase c-fos mRNA in PC12 cells that were depleted of protein kinase C by a 24-h treatment with phorbol 12,13-dibutyrate or incubated with the protein kinase C inhibitor H-7. In contrast, an inhibitor of calmodulin-dependent protein kinase, KN-62, and an inhibitor of calmodulin, W-7, did not block the induction of c-fos mRNA by lead. An increase in serum-response element DNA-binding activity was observed in nuclear extracts from PC12 cells exposed to lead. It is interesting that lead activated protein kinase C isoforms delta and epsilon, but not isoforms alpha and beta. In conclusion, lead appears to induce the expression of immediate early genes by a mechanism that requires protein kinase C.