Phorbol ester stimulates ethanolamine release from the metastatic basal prostate cancer cell line PC3 but not from prostate epithelial cell lines LNCaP and P4E6.

BACKGROUND: Malignancy alters cellular complex lipid metabolism and membrane lipid composition and turnover. Here, we investigated whether tumorigenesis in cancer-derived prostate epithelial cell lines influences protein kinase C-linked turnover of ethanolamine phosphoglycerides (EtnPGs) and alters the pattern of ethanolamine (Etn) metabolites released to the medium. METHODS: Prostate epithelial cell lines P4E6, LNCaP and PC3 were models of prostate cancer (PCa). PNT2C2 and PNT1A were models of benign prostate epithelia. Cellular EtnPGs were labelled with [1-(3)H]-Etn hydrochloride. PKC was activated with phorbol ester (TPA) and inhibited with Ro31-8220 and GF109203X. D609 was used to inhibit PLD (phospholipase D). [(3)H]-labelled Etn metabolites were resolved by ion-exchange chromatography. Sodium oleate and mastoparan were tested as activators of PLD2. Phospholipase D activity was measured by a transphosphatidylation reaction. Cells were treated with ionomycin to raise intracellular Ca(2+) levels. RESULTS: Unstimulated cell lines release mainly Etn and glycerylphosphorylEtn (GPEtn) to the medium. Phorbol ester treatment over 3h increased Etn metabolite release from the metastatic PC3 cell line and the benign cell lines PNT2C2 and PNT1A but not from the tumour-derived cell lines P4E6 and LNCaP; this effect was blocked by Ro31-8220 and GF109203X as well as by D609, which inhibited PLD in a transphosphatidylation reaction. Only metastatic PC3 cells specifically upregulated Etn release in response to TPA treatment. Oleate and mastoparan increased GPEtn release from all cell lines at the expense of Etn. Ionomycin stimulated GPEtn release from benign PNT2C2 cells but not from cancer-derived cell lines P4E6 or PC3. Ethanolamine did not stimulate the proliferation of LNCaP or PC3 cell lines but decreased the uptake of choline (Cho). CONCLUSIONS: Only the metastatic basal PC3 cell line specifically increased the release of Etn on TPA treatment most probably by PKC activation of PLD1 and increased turnover of EtnPGs. The phosphatidic acid formed will maintain a cancer phenotype through the regulation of mTOR. Ethanolamine released from cells may reduce Cho uptake, regulating the membrane PtdEtn:PtdCho ratio and influencing the action of PtdEtn-binding proteins such as RKIP and the anti-apoptotic hPEBP4. The work highlights a difference between LNCaP cells used as a model of androgen-dependent early stage PCa and androgen-independent PC3 cells used to model later refractory stage disease.

Immunocytochemical characterisation of cell cultures grown from dissociated 1-2-day post-natal rat cerebral tissue. A developmental study.

A range of cell-specific markers have been employed with immunocytochemical methods to characterise and quantitate the cell types present in mixed brain cell cultures derived from dissociated 1-2-day post-natal rat cerebral hemispheres and grown in the presence of FCS. Protoplasmic astrocytes (GFAP+, A2B5-) were the major cell type to develop in culture, a confluent monolayer forming in 5-8 days. A population of smaller round cells of oligodendrocyte-like morphology appeared on this astrocyte layer. Greater than 70% of these smaller cells were GC- and thus were not oligodendrocytes. The GC- cells were A2B5+ and, in early cultures, may therefore be progenitor glial cells. Examination of GFAP and A2B5 co-expression by these smaller cells was difficult due to the dense underlying GFAP+ astrocyte layer. In less dense areas of older cultures these smaller cells with processes were GFAP+ and A2B5+: these are Type 2, fibrous astrocytes. GC+ oligodendrocytes, comprising 5-10% of the total identified cell population, were initially distributed over the astrocyte monolayer; in older cultures (after about 8 days) GC+ cells were observed in clumps over places where NF+ cells were identifiable. Such GC+ cells mostly became MBP+. Neurones accounted for about 6% of the identifiable cells in early cultures but a lower percentage in older cultures. Minor populations of ependymal cells and macrophages were present; cells displaying fibronectin, fibroblasts, were rarely identified. Use of horse serum in place of FCS gave lower yields of GC+ cells in cultures, slowed down astrocyte development, and resulted in the formation of trunks of GFAP+ cells throughout cultures. Other sera gave lower numbers of GC+ cells.

Immunocytochemical demonstration of glial-neuronal interactions and myelinogenesis in subcultures of rat brain cells.

Subcultures have been established from primary rat brain cell cultures and have been characterised with a range of cell-specific immunocytochemical markers. The subcultures are mainly composed of fibrous astrocytes, oligodendrocytes and neurones. The cells do not divide to any great extent giving a system where it is possible to follow culture development at the cellular level for a number of weeks. During this time oligodendrocytes colonise subpopulations of neurones, differentiate further showing the presence of myelin basic protein and elaborate myelin-like membrane; the fibrous astrocytes remain scattered uniformly throughout the cultures. Radially oriented processes emerge from the oligodendrocyte-neurone aggregates which subsequently coalesce to form fascicles that link the clusters of cells together. These fascicles react with antibodies for both neurofilament protein and myelin basic protein. The subcultures provide a straightforward system that is composed of cells derived entirely from the CNS, is free from mitotic inhibitors and yet retains a sufficiently low cell density to allow immunocytochemical identification of the cell types present. The subcultures should be useful for the study of trophic interactions between oligodendrocytes and neurones as well as the early events associated with myelinogenesis.

Chronic relapsing experimental allergic encephalomyelitis. Immunological and blood--cerebrospinal fluid barrier-dependent changes in the cerebrospinal fluid.

Cerebrospinal fluid (CSF) and plasma were taken from strain 13 guinea pigs in various stages of chronic relapsing experimental allergic encephalomyelitis using techniques which allowed repeated sampling from the same animal. Samples were assayed for albumin and IgG and the corresponding CSF/plasma quotients evaluated graphically using a method which could discriminate between blood-CSF barrier dysfunction and local IgG synthesis in the central nervous system (CNS). During the disease a 2-3-fold increase in plasma IgG concentration developed and an increase in blood-CSF permeability was noted. Isoelectric focusing revealed an oligoclonal IgG pattern identical in both plasma and CSF. The results provided no evidence for a local production of IgG in the CNS like that which is known to occur in multiple sclerosis.

Chronic relapsing experimental allergic encephalomyelitis. The presence in the cerebrospinal fluid of factors chemotactic for monocytes.

Cerebrospinal fluid (CSF) was removed from guinea pigs with chronic relapsing experimental allergic encephalomyelitis (CR-EAE) and control inoculated animals by puncture of the cisterna magna. The fluid from 7/8 animals in relapse and 2/4 animals in remission phases of CR-EAE was found to promote the migration of peripheral blood monocytes through a 5-micron pore diameter polycarbonate membrane filter. Monocytes were also found to orient towards the migration-stimulating CSF in a gradient formed between such fluid and CSF derived from a control animal, thereby indicating the presence of a chemotactic factor. The factor responsible for promoting monocyte migration had a molecular weight of between 50 000 and 300 000 as defined by ultrafiltration. The results are discussed in relation to the known pathohistological features of the chronic relapsing disease.

Quantitative analysis of the cellular constituents of the cerebrospinal fluid in chronic relapsing experimental allergic encephalomyelitis.

Cerebrospinal fluid (CSF) was taken from guinea pigs in various stages of chronic relapsing experimental allergic encephalomyelitis (CR-EAE). The leucocytes in CSF samples were counted and subjected to immunocytochemical analysis using monoclonal antibodies selectively recognising guinea pig T cells, macrophages or Ia antigens. The results showed that total leucocyte numbers and the proportion of macrophages in CSF were elevated in the acute phase of CR-EAE but samples of CSF from animals in early relapse did not show a significant elevation in leucocyte count or macrophage content. In addition the level of T cell activation was higher in CSF than in peripheral blood during disease and was highest in the acute and immediately post-acute phases of the CR-EAE.

Lymphocytic activation in peripheral blood and cerebrospinal fluid during the course of chronic relapsing experimental allergic encephalomyelitis.

A monoclonal antibody against the human interleukin-2 receptor (anti-Tac) has been found to cross-react with an antigen on the surface of guinea pig leucocytes. Cells marking with anti-Tac and with an anti-pan T cell monoclonal antibody have been quantitated in the peripheral blood and cerebrospinal fluid (CSF) of guinea pigs with chronic relapsing experimental allergic encephalomyelitis (CR-EAE). T cells account for about 90% of peripheral blood leucocytes in all animals whilst in the CSF, T cells are the major contributor only when there is a pleocytosis. The proportion of T cells marking with anti-Tac, a measure of T cell activation, in blood and CSF of control animals is 12%, rising to 23% in blood in the post-acute phase of the disease. However, a fall in the blood Tac/T ratio to 13% occurs during the first 10 days of relapse with a subsequent rise to 30-35%. This change is related to the time after onset of relapse irrespective of the subsequent course of the disease. From first relapse onwards CSF lymphocytes show a greater level of activation than lymphocytes from paired peripheral blood samples but the proportion of Tac+ cells in CSF does not increase with increasing CSF pleocytosis. The data is consistent with migration of activated T cells from blood to CSF at the onset of relapse.

Chronic relapsing experimental allergic encephalomyelitis. Inhibition of lymphocyte mitogenesis by disease-related serum factors.

Peripheral blood mononuclear cells (PBMC) from strain 13 guinea pigs at various stages of chronic relapsing experimental allergic encephalomyelitis (CREAE) showed significantly reduced reactivity to the polyclonal T cell mitogens, phytohaemagglutinin and concanavalin A. Serum taken at the same time revealed an increase in inhibitors of lymphocyte mitogenesis. The factors identified appeared to inhibit the initial stages of lymphocyte proliferation: they were not cytotoxic for lymphocytes nor were they complement-dependent.

Monensin and verapamil do not alter intracellular localisation of daunorubicin in multidrug resistant human KB cells.

The effects of monensin, verapamil and several inhibitors of membrane transport processes on the accumulation of [3H] daunorubicin by human KB-A1 cells have been investigated to determine the role of subcellular vesicular transport in the multidrug resistance phenotype. The Golgi inhibitor, brefeldin A, had no effect on drug accumulation, which suggests that vesicular transport is not a significant factor in drug resistance in these cells. KB-A1 cells were collaterally sensitive to both monensin and verapamil. Both of these compounds reduced drug efflux but did not alter subcellular distribution of daunorubicin, consistent with the view that monensin, like verapamil, acts directly on P-glycoprotein.

The origin and specificity of intrathecal IgG in chronic relapsing experimental allergic encephalomyelitis.

The source of IgG in the cerebrospinal fluid (CSF) in guinea pigs with chronic relapsing experimental allergic encephalomyelitis (CR-EAE) was investigated using quotient analysis of total IgG and albumin concentrations and by computing CSF-plasma ratios of specific IgG concentrations. Increased blood-CSF barrier (B-CSFB) permeability was shown by elevated albumin quotients in both relapse and remission phases of CR-EAE and intrathecal production of IgG was indicated by raised ratios of IgG to albumin in the CSF. Intrathecal IgG synthesis was greatest in guinea pigs which had little B-CSFB damage. When enzyme-linked immunosorbent assays (ELISA) for whole cord, myelin basic protein (MBP) or Mycobacterium tuberculosis were performed with CSF and plasma adjusted to the concentration of total IgG, the CSF/plasma ratios of ELISA results for specific antibodies were less then unity and ratios for whole cord and MBP were lower than those for M. tuberculosis. There was thus no evidence for a selective increase in the CSF of antibody specific either for the neuroantigens tested or for adjuvant components. The CSF-plasma ratios for each specific antibody were inversely correlated with the extent of total IgG intrathecal synthesis, suggesting that much of the antibody production within the CNS is the result of polyclonal B cell activation.

The induction of liposome aggregation by myelin basic protein.

Myelin basic protein derived from bovine spinal cord has been interacted with liposomes of varying brain lipid compositions. The effects of salt and protein concentration on liposome cross linking has been investigated. It appears that myelin basic protein cannot link liposomes composed of brain-derived phosphatidyl choline. Myelin basic protein can link liposomes composed of phosphatidyl serine; phosphatidyl serine + cholesterol; phosphatidyl serine + cholesterol + cerebroside sulphate. Linking of liposomes occurs at protein concentrations lower than those required for myelin basic protein dimers to be formed. Therefore, it seems that the monomeric form of myelin basic protein links lipid bilayers. The presence of cholesterol in the bilayer increases the ability of myelin basic protein to aggregate such liposomes compared with the linking ability of the polycationic polypeptide, poly-l-lysine.

Galactosyl ceramides of the myelin sheath: thermal studies.

The thermal properties of several hydrated glycosyl ceramides have been investigated by differential scanning calorimetry in relation to the role of galactosyl ceramides in the myelin sheath of central nerve tissue. Myelin galactosyl ceramides are characterised by the presence of a large proportion of alpha-hydroxy fatty acids which lower the phase transition of this lipid class in comparison to galactosyl ceramides with normal fatty acids of similar chain length. Alteration of the sugar headgroup from galactose to glucose produced no significant change in the transition temperature of the lipid. The presence of a disaccharide headgroup lowered the transition temperature and altered the structure of the endotherm. Increasing concentrations of cholesterol in aqueous dispersions of galactosyl ceramide progressively decreased the enthalpy of the phase transition until, in the presence of 34 mole % cholesterol, it was no longer detectable. Examination of the effects of a range of phospholipids on the phase transition of galactosyl ceramides suggest that the phase behaviour of phospholipid-galactosyl ceramide mixtures is complex. However, only a single endotherm was observed in all the phospholipid-galactosyl ceramide systems examined. This tends to indicate that segregated phases enriched in galactosyl ceramides do not occur in the intact myelin membrane.

Susceptibility of myelin glycerophospholipids and sphingolipids to oxidative attack by hydroxyl free radicals as measured by the thiobarbituric acid test.

Glycerophospholipids and sphingolipids typical of the myelin sheath have been examined for the formation of thiobarbituric acid reactive materials after attack by hydroxyl free radicals. Thiobarbituric acid (TBA) reactive materials are formed following exposure of phosphatidylcholine (PC), phosphatidylserine (PS) and phosphatidylethanolamine (PE) to hydroxyl radical attack in the order PS > PC > PE while the sphingolipids, sphingomyelin and cerebroside and the sterol cholesterol, did not form TBA reactive materials. Inclusion of cholesterol into PC vesicles reduced the formation of TBA reactive materials on hydroxyl radical attack while with PS there was an increase. In mixed glycerophospholipid systems inclusion of cerebroside reduced formation of TBA reactive materials following hydroxyl radical attack on phosphatidylcholine and phosphatidylserine, but not phosphatidylethanolamine. The findings are discussed in relation to the oxidative damage we have observed in the central nervous system at different stages of chronic relapsing experimental allergic encephalomyelitis [Brett R. and Rumsby M. G. Neurochem. Int. 23, 35-44 (1993)].

Evidence of free radical damage in the central nervous system of guinea-pigs at the prolonged acute and early relapse stages of chronic relapsing experimental allergic encephalomyelitis.

Central nervous system tissue from guinea-pigs in various stages of chronic relapsing experimental allergic encephalomyelitis (CR-EAE) has been analysed for evidence of free radical damage using the thiobarbituric acid test as an indicator of oxidative changes. Levels of thiobarbituric acid-reactive materials in brain and spinal cord regions from prolonged acute and early relapse phases of CR-EAE were significantly higher than in central nervous system tissue from Freund's adjuvant control and normal uninoculated animals. In the late relapse phase of CR-EAE, levels of thiobarbituric acid-reactive materials in CNS samples were at control values. Fluorescence analysis of central nervous system tissue confirmed evidence of increased free radical damage in prolonged acute and early relapse stages of CR-EAE. The increased oxidative damage observed in the central nervous system in CR-EAE is discussed in relation to the role of macrophages and their oxidative burst.

Action of phospholipase C (Bacillus cereus) on isolated myelin sheath preparations.

The action of phospholipase C (Bacillus cereus) on the phospholipids of myelin sheath preparations has been investigated. With freshly isolated bovine brain myelin about 40% of the total phospholipid could be hydrolyzed by this enzyme. With bovine spinal cord myelin the phospholipid seemed more resistant to attack whereas the opposite was the case with myelin from guinea-pig brain or rat brain. With fresh bovine brain myelin, phosphatidylcholine and the ethanolamine-containing phospholipids were the main targets for the enzyme with lesser extents of hydrolysis occurring with phosphatidylserine and sphingomyelin. The effect of exposing bovine brain myelin to structural perturbants prior to enzyme digestion indicated that trypsin pretreatment had no significant effect, whereas marked enhancement of the extent of phospholipid hydrolysis occurred following lyophilization + rehydration, or pretreatment of myelin with HCl, Triton TX-100/ammonium acetate or deoxycholate. The effect of myelin pretreatment on the degradation of the individual phospholipid classes was also studied.

On the disposition of phospholipids in freshly isolated myelin sheath preparations from bovine brain.

The extent of availability of the four major classes of phospholipids in freshly isolated bovine brain myelin has been studied using phospholipase C (Clostridium perfringens), phospholipase A (bee venom), sphingomyelase (Staphalococcus aureus) and trinitrobenzenesulphonate. The results have been compared with those of a previous study (Gwarsha et al., 1984). Taken together, the data suggest that 47-67% of the phosphatidylcholine, 45-66% of the ethanolamine-containing phosphoglycerides, 52-55% of the spingomyelin and 10-32% of the phosphatidylserine + phosphatidylinositol in the fresh myelin is available to these probes and hence may be located at the external surface of the membrane bilayer.

The protein kinase C, calcium-independent and calcium-dependent kinase activities of glial cells in primary culture and subculture.

The activity of calcium-independent, calcium-dependent and calcium + phospholipid-dependent (protein kinase C) kinases in cytosol fractions from rat brain glial cells in primary culture and from subcultured astrocytes and in oligodendrocyte-type 2 astrocyte lineage glia has been measured with histone Type IIIS as substrate. Accurate measurement of protein kinase C activity was achieved only after chromatography of glial cytosols on DE-52 anion exchange columns to remove an endogenous inhibitor, identified tentatively as a phosphatase possibly of the protein phosphatase 2A class. The specific activity of protein kinase C in glial cell cytosol increased from 7.5 +/- 0.8 to 37.9 +/- 0.9 nmol32P incorporated/mg protein/10 min with increasing culture age. Protein kinase C activity in glial cytosol was significantly higher when primary cultures were grown in a defined medium lacking serum. Astrocyte-conditioned medium and phorbol esters caused a rapid translocation of glial cell protein kinase C activity from cytosol to membrane compartments. Myelin basic protein and protamine have been compared with histone as substrates for measurement of calcium-independent, calcium-dependent and calcium + phospholipid-dependent kinase activities in glial cytosol.

A syringe modification for the simple and rapid dissociation of post-natal rat cerebral tissue for preparing primary cultures of mixed glial cells.

Mechanical disruption of 2-day post-natal rat cerebral tissue without enzyme pretreatment through 21-gauge and 23-gauge hypodermic needles on a 1 ml disposable plastic syringe, gives dissociated preparations which contain between 1.7 and 2.1 X 10(7) cells/100 mg wet tissue. At a plating density of between 7.1 and 9.2 X 10(5)/cm2 in culture, between 2 X 10(5) and 2 X 10(6) cells are recovered after 7 days. Use of needles finer than 23-gauge resulted in lower cell yields and loss of galactocerebroside-positive oligodendrocytes. The method described is convenient for the routine preparation of mixed primary glial cultures on a regular basis where many cerebra need to be processed.

Protein kinase C activity in soluble fractions from glial cells in primary culture and subcultures.

Protein kinase C (calcium + phospholipid-dependent kinase) activity has been measured in soluble 100,000 g fractions from mixed glial cells in primary culture; in 12 day cultures the specific activity (mean +/- S.D.) was 184 +/- 10 pmol 32P incorporated/10 min/mg protein. In glial cell subcultures lacking protoplasmic astrocytes protein kinase C specific activity was lower. An inhibitor of protein kinase C in 100,000 g supernatants was removed by chromatography through DE-52 anion exchange resin increasing the specific activity of the calcium + phospholipid-dependent kinase about 20 times. Protein kinase C was also associated with membrane fractions from glial cells; the membrane-associated enzyme had a higher specific activity than in the cytoplasm.

Stimulation of protein phosphorylation in mixed glial cell primary cultures and subcultures by the phorbol ester 12-O-tetradecanoylphorbol-13-acetate.

Glial cell primary cultures consisting of protoplasmic and fibrous astrocytes, oligodendrocytes and progenitor glial cells incubated in medium containing 0.5% foetal calf serum and treated with 25 nM 12-o-tetradecanoylphorbol-13-acetate (TPA) for periods between 15 and 60 min showed a stimulation of protein phosphorylation which was most prominent in a polypeptide with a molecular weight of about 80,000 Da. Glial subcultures consisting mainly of Type 2 astrocytes, oligodendrocytes and progenitor glia showed a similar TPA stimulation of 80,000 Da protein phosphorylation detectable within 1 min of phorbol ester addition. TPA treatment of primary glial cultures led to an enhancement of phospholipid turnover but exposure of primary glial cultures to concentrations of TPA up to 250 nM caused no morphological change in protoplasmic astrocytes. 4-Phorbol (4-PH) or dimethylsulfoxide (DMSO) was without effect on protein phosphorylation or lipid turnover in glial cultures.