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.

Human prostate cell lines from normal and tumourigenic epithelia differ in the pattern and control of choline lipid headgroups released into the medium on stimulation of protein kinase C.

BACKGROUND: Expression of protein kinase C alpha (PKCα) is elevated in prostate cancer (PCa); thus, we have studied whether the development of tumourigenesis in prostate epithelial cell lines modifies the normal pattern of choline (Cho) metabolite release on PKC activation. METHODS: Normal and tumourigenic human prostate epithelial cell lines were incubated with [(3)H]-Cho to label choline phospholipids. Protein kinase C was activated with phorbol ester and blocked with inhibitors. Choline metabolites were resolved by ion-exchange chromatography. Phospholipase D (PLD) activity was measured by transphosphatidylation. Protein expression was detected by western blotting and/or RT-PCR. Choline uptake was measured on cells in monolayers over 60 min. RESULTS: Normal prostate epithelial cell lines principally released phosphocholine (PCho) in contrast to tumourigenic lines, which released Cho. In addition, only with normal cell lines did PKC activation stimulate Cho metabolite release. Protein kinase C alpha expression varied between normal and tumourigenic cell lines but all showed a PKCα link to myristoylated alanine-rich C kinase substrate (MARCKS) protein. The five cell lines differed in Cho uptake levels, with normal PNT2C2 line cells showing highest uptake over 60 min incubation. Normal and tumourigenic cell lines expressed mRNA for PLD1 and PLD2, and showed similar levels of basal and PKC-activated PLD activity. CONCLUSIONS: The transition to tumourigenesis in prostate epithelial cell lines results in major changes to Cho metabolite release into the medium and PKC signalling to phosphatidylcholine turnover. The changes, which reflect the metabolic and proliferative needs of tumourigenic cells compared with untransformed cells, could be significant for both diagnosis and treatment.

Differentiation of bipolar CG-4 line oligodendrocytes is associated with regulation of CREB, MAP kinase and PKC signalling pathways.

Undifferentiated bipolar CG-4 cell line oligodendrocytes provide a model system for the O-2A progenitor cell from which oligodendrocytes are derived both in vivo and in vitro. The exchange of neuroblastoma conditioned basal media for basal media causes differentiation of undifferentiated bipolar CG-4 cells into multipolar oligodendrocyte-like cells whilst replacement with basal media containing 20% foetal bovine serum favours the formation of type-2 astrocyte-like cells. Here, we demonstrate that activation of these differentiation pathways correlates with distinct changes both in cell metabolism and in signal transduction. Exchange of neuroblastoma conditioned media for basal media correlates with stimulation of basal metabolic activity, reduced phosphorylation of p44/42 MAP kinase and reduced phosphorylation of the transcription factor CREB. In contrast, differentiation with basal medium containing 20% foetal bovine serum (FBS), into type 2 astrocyte-like cells, correlates with reduction in basal metabolic activity, increased phosphorylation of p44/42 MAP kinase and increased phosphorylation of the transcription factor CREB. Inhibition of protein kinase C blocked both the metabolic and morphological changes associated with differentiation towards mature multipolar oligodendrocyte-like cells. Inhibition of PKA and MEK did not effect metabolic activity. The rapid return of neuroblastoma conditioned basal media to cells treated with basal media, increased phosphorylation of CREB and MAP kinase. These results demonstrate that protein kinase C and p44/42 MAP kinase signalling pathways are modulated during bipolar CG-4 cell differentiation and demonstrate that the transcription factor CREB may play a pivotal role in differentiation along oligodendrocyte-or astrocyte-lineages.

The 5' UTR of protein kinase C epsilon confers translational regulation in vitro and in vivo.

We have examined translational regulation conferred by the 5' untranslated region (UTR) of PKCepsilon on expression of the luciferase reporter gene in vitro, using rabbit reticulocyte lysates and in vivo, in contact-inhibiting mouse Swiss 3T3 fibroblasts and non-contact-inhibiting Swiss 3T6 fibroblasts. In rabbit reticulocyte lysates, the 5' UTR of PKCepsilon significantly represses translation. In 3T3 and 3T6 cells, the 5' UTR of PKCepsilon reduces luciferase activity, but not to the same extent as it does in vitro. In rabbit reticulocyte lysate, the degree of repression mediated by different PKCepsilon 5' UTR-deletion constructs correlates with the free energy (DeltaG) of their predicted secondary structures. However, in cells, secondary structure is not the only determinant of repression; an internal region of the 5' UTR is both necessary and sufficient for repression. Mutation of an upstream AUG (uAUG) motif in this region partially relieves repression. We conclude that the 5' UTR of PKCepsilon can mediate translational regulation and that translation inhibition in vivo involves the uAUG motif. Our findings also suggest that there are factors present in fibroblasts, but not in rabbit reticulocyte lysates that substantially overcome the repressive qualities of the long, structured 5' UTR. Thus, we have identified a potential new level of regulation of PKC in mammalian cells.

Protein kinase C activation by 12-0-tetradecanoylphorbol 13-acetate in CG-4 line oligodendrocytes stimulates turnover of choline and ethanolamine phospholipids by phospholipase D and induces rapid process contraction.

Treatment of [3H]-choline- or [14C]-ethanolamine-labelled undifferentiated bipolar and differentiated multipolar CG-4 line oligodendrocytes with 12-0-tetradecanoylphorbol 13-acetate (TPA) to activate protein kinase C stimulated the release of choline or ethanolamine metabolites to the medium over controls. Ro31-8220, a PKC inhibitor, reduced TPA-stimulated release of choline- and ethanolamine-metabolites to basal levels. TPA treatment of both bipolar and multipolar cells caused rapid contraction of processes leaving rounded up cells: this effect was blocked by Ro31-8220. After 12-15 h exposure to TPA, bipolar undifferentiated CG-4 line cells extended short processes again and the cells became multipolar. Nocodozole, an agent which disrupts microtubules and caused CG-4 line cells to round up, caused increased choline or ethanolamine-metabolite release to the medium over basal levels suggesting that some release during TPA-treatment might occur due to process fragmentation. However, the transphosphatidylation reaction confirmed that phospholipase D was active in these cells. Exposure of bipolar undifferentiated CG-4 line cells to TPA resulted in down-regulatation of PKC-alpha and PKC-beta which could not be detected by Western blotting after a few hours; PKC-epsilon was down-regulated much more slowly but PKCs delta, zeta and iota were not influenced by 48 h exposure of cells to TPA. Formation of phosphatidylethanol in the transphosphatidylation reaction was markedly reduced in TPA down-regulated cells indicating a role for PKCs alpha and beta in phospholipase D activation in CG-4 line oligodendrocytes.

Signalling pathways regulating the dephosphorylation of Ser729 in the hydrophobic domain of protein kinase Cepsilon upon cell passage.

We have recently demonstrated that in quiescent fibroblasts protein kinase C (PKC) epsilon(95) is phosphorylated at Ser(729), Ser(703), and Thr(566) and that upon passage of quiescent cells phosphorylation at Ser(729) is lost, giving rise to PKCepsilon(87). Ser(729) may be rephosphorylated later, suggesting cycling between PKCepsilon(87) and PKCepsilon(95). Here we show that the dephosphorylation at Ser(729) is insensitive to okadaic acid, calyculin, ascomycin C, and cyclosporin A, suggesting that dephosphorylation at this site is not mediated through protein phosphatases 1, 2A or 2B. We demonstrate that this dephosphorylation at Ser(729) requires serum and cell readhesion and is sensitive to rapamycin, PD98059, chelerythrine, and Ro-31-8220. These results suggest that the phosphorylation status of Ser(729) in the hydrophobic domain at Ser(729) is regulated independently of the phosphorylation status of other sites in PKCepsilon, by a mTOR-sensitive phosphatase. The mitogen-activated protein kinase pathway and PKC are also implicated in regulating the dephosphorylation at Ser(729).

Changes in protein kinase C epsilon phosphorylation status and intracellular localization as 3T3 and 3T6 fibroblasts grow to confluency and quiescence: a role for phosphorylation at ser-729?

Protein kinase C (PKC) epsilon in 3T3 and 3T6 fibroblasts and in C6 glioma cells migrated on SDS/PAGE predominantly as a doublet with molecular masses of 87 and 95 kDa (PKC epsilon(87) and PKC epsilon(95) respectively). PKC epsilon(95) predominates when cells reach confluency but PKC epsilon(87) was the main form detected within 15 min when confluent cells were passaged at low cell density into fresh medium containing serum and allowed to adhere. Matrix-assisted laser-desorption ionization-time-of-flight MS analysis and experiments with phosphospecific antibodies revealed that PKC epsilon(87) is phosphorylated at Thr-566 and Ser-703, and PKC epsilon(95) is additionally phosphorylated at Ser-729. Cell fractionation studies revealed that PKC epsilon(95) is associated with the nuclear fraction, whereas PKC epsilon(87) was found in the 100,000 g cytosol fraction. Immunofluorescence studies confirmed these findings and showed that PKC epsilon(95) had a perinuclear, probably Golgi, localization and PKC epsilon(87) was distributed in the cytosol. It is proposed that phosphorylation at Ser-729 may be important for determining the intracellular localization of PKC epsilon, and that a specific Ser-729 phosphatase may be activated on cell passage to convert PKC epsilon(95) to PKC epsilon(87).

Phage display identifies thioredoxin and superoxide dismutase as novel protein kinase C-interacting proteins: thioredoxin inhibits protein kinase C-mediated phosphorylation of histone.

Using phage display we identify the redox proteins thioredoxin and superoxide dismutase (SOD) as novel protein kinase C (PKC)-interacting proteins. Overlay assays demonstrated that PKC bound to immobilized thioredoxin, providing supporting evidence for the phage display results. Kinase assays demonstrated that SOD and thioredoxin were not direct substrates for PKC but that both proteins blocked autophosphorylation of PKC. Moreover, thioredoxin inhibited PKC-mediated phosphorylation of histone (IC(50) of approx. 20 ng/ml).

Substratum of pleiotrophin (HB-GAM) stimulates rat CG-4 line oligodendrocytes to adopt a bipolar morphology and disperse: primary O-2A progenitor glial cells disperse similarly on pleiotrophin.

Pleiotrophin (HB-GAM), an extracellular matrix-associated protein with a high content of basic amino acid residues, is expressed in the central nervous system during late pre- and early post-natal development and promotes neurite outgrowth in vitro. Here, we show that, on a substratum of pleiotrophin formed from a 5 or 10 microg/ml solution, undifferentiated rat CG-4 line oligodendrocytes adopt a bipolar morphology and disperse over the substratum, as we have previously shown with poly-L-lysine (Rumsby et al. Neurosci. Res. Commun. 23:101-109, 1998). On pleiotrophin substrata formed from coating solutions of 1 microg/ml and below, CG-4 line cells form aggregates and do not disperse, as is also the case with poly-L-lysine. The same dispersing effect is observed with rat primary 0-2A progenitor glial cells on pleiotrophin substrata from solutions of 5 and 10 microg/ml: 0-2A cells aggregate together on pleiotrophin substrata formed from lower concentrations and do not disperse. A pleiotrophin substratum enhances proliferation of CG-4 line oligodendrocytes and primary 0-2A progenitor glial cells. The results show that pleiotrophin provides a substratum that can influence progenitor oligodendrocyte morphology, aid cell dispersion, and perhaps also enhance progenitor oligodendrocyte cell growth.

The bisindolylmaleimide protein kinase C inhibitor, Ro 32-2241, reverses multidrug resistance in KB tumour cells.

Ro 32-2241 is a bisindolylmaleimide that selectively inhibits protein kinase C (PKC) as compared with other protein kinases. Experiments were carried out to examine its potential as a multidrug resistance-reversing agent. Ro 32-2241 inhibited efflux, and increased accumulation, of [3H]-daunomycin in multidrug-resistant (MDR) KB-8-5 and KB-8-5-11 cells and had no effect on drug-sensitive KB-3-1 cells. Ro 32-2241 completely reversed the doxorubicin resistance of KB-8-5 and KB-8-5-11 cells, showing no effect on the sensitivity of drug-sensitive KB-3-1 cells. The potency of Ro 32-2241 was comparable with that of cyclosporin A and better than that of verapamil, known modulators of multidrug resistance. Ro 32-2241 also completely reversed the taxol resistance of KB-8-5 cells and partially reversed the resistance of KB-8-5-11 cells. Vinblastine resistance was also partially reversed. Mechanistic experiments were carried out to determine whether Ro 32-2241 interacted with P-glycoprotein (Pgp) directly. Increased efflux of [14C]-Ro 32-2241 was seen with the more resistant KB-8-5-11 cells (although the percentage effluxed was very low as compared with [3H]-daunomycin), suggesting that Ro 32-2241 can act as a substrate for Pgp. Direct interaction of Ro 32-2241 with Pgp was confirmed by demonstration that it inhibited binding of [3H]-azidopine to Pgp in KB-8-5-11 membranes. In conclusion, Ro 32-2241, acting directly on Pgp (rather than, or in addition to, an effect on PKC), is effective in reducing or reversing resistance to doxorubicin, taxol and vinblastine in human tumour cells with a clinically relevant degree of MDR. However, results of in vivo experiments conducted to investigate the effects of Ro 32-2241 on resistance to doxorubicin suggest that it may not be possible to achieve sufficiently high levels of Ro 32-2241 in vivo to modulate MDR.

Rat oligodendrocyte O-2A precursor cells and the CG-4 oligodendrocyte precursor cell line express cadherins, beta-catenin and the neural cell adhesion molecule, NCAM.

Oligodendrocyte precursor cell migration throughout the developing central nervous system (CNS) and cessation of migration are poorly understood but are likely to involve cell adhesion molecules. The expression and distribution of neural cell adhesion molecule (NCAM), cadherins and beta-catenin were investigated in the CG-4 cell line and primary rat oligodendrocyte progenitor cells (O-2A) by immunofluorescence and Western blotting. NCAM was expressed by both cell types and was found all over the surface of both CG-4 cells and O-2A progenitor glia. The presence of a cadherin was detected in both CG-4 cells and O-2A progenitor glia, and this molecule was distributed all over the cell body and cell processes at different stages of differentiation. Beta-catenin showed a very similar distribution to that of the cadherin. We conclude that CG-4 cells are a valid model system to study cell adhesion molecule expression and function in oligodendrocyte progenitor cells.

Protein kinases and multidrug resistance.

The role of protein kinases in the multidrug resistance phenotype of cancer cell lines is discussed with an emphasis on protein kinase C and protein kinase A. Evidence that P-glycoprotein is phosphorylated by these kinases is summarised and the relationship between P-glycoprotein phosphorylation and the multidrug-resistant phenotype discussed. Results showing that protein kinase C, particularly the alpha subspecies, is overexpressed in many MDR cell lines are described: this common but by no means universal finding seems to be drug- and cell line-dependent and in only in a few cases is there a direct correlation between protein kinase C activity and multidrug resistance. From co-immunoprecipitation results it is suggested that P-glycoprotein is a specific protein kinase C receptor, as well as being a substrate. Revertant experiments provide conflicting results as to a direct relationship between expression of P-glycoprotein and protein kinase C. Evidence that protein kinase A influences P-glycoprotein expression at the gene level is well documented and the mechanisms by which this occurs are becoming clarified. Results on the relationship between protein kinase C and multidrug resistance using many inhibitors and phorbol esters are difficult to interpret because such compounds bind to P-glycoprotein. In spite of huge effort, a direct involvement of protein kinase C in regulating multidrug resistance has not yet been firmly established. However, evidence that PKC regulates a Pgp-independent mechanism of drug resistance is accumulating.

Ro31-8220 inhibits protein kinase C to block the phorbol ester-stimulated release of choline- and ethanolamine-metabolites from C6 glioma cells: p70 S6 kinase and MAPKAP kinase-1beta do not function downstream of PKC in activating PLD.

The use of bisindolylmaleimide derivatives of staurosporine as selective inhibitors of protein kinase C (PKC) is in doubt following the report by Alessi [FEBS Lett. 402 (1997) 121-123] that Ro31-8220 and GF109203X are potent in vitro inhibitors of p70 S6 kinase and mitogen-activated protein kinase-activated protein kinase-1beta, as well as of PKC. Here we show that the phorbol ester-stimulated release of choline- and ethanolamine-metabolites from C6 glioma cells due to phospholipid hydrolysis by phospholipase D (PLD) is not inhibited by rapamycin or PD98059, specific inhibitors respectively of p70 S6 kinase and MAPKK (MEK) and thus of MAPKAP kinase-1beta but is still completely blocked by Ro31-8220. We conclude therefore that p70S6k and MAPKAP kinase-1beta as well as MAPK are not involved in signalling pathways downstream of PKC that regulate phorbol ester-stimulated phospholipid turnover and that the inhibitory action of Ro31-8220 occurs by blocking PKC which regulates at least one pathway to PLD activation. The PI-3 kinase inhibitor, wortmannin, inhibits the phorbol ester-stimulated release of ethanolamine- but not choline-metabolites from C6 cells suggesting that different PLD isoforms regulate the turnover of PtdEth and PtdCho in C6 cells. Both PLD isoforms are activated via PKC but the PtdEth-PLD is also regulated via a wortmannin-sensitive pathway.

Protein expression of the epsilon subspecies of protein kinase C ceases as Swiss 3T6 fibroblasts increase in cell density even though message for the protein is still present.

We have noted previously that growth of C6 glioma cells from low cell density to confluency and quiescence in serum is accompanied by changes in protein content of different protein kinase C (PKC) subspecies. Here we show that the same occurs as non-contact-inhibiting Swiss 3T6 fibroblasts grow to high density in the presence of serum. Protein expression of PKC subspecies alpha and delta increases as the cells increase in density while that of PKC-zeta remains the same. Unusually, protein expression of PKC-epsilon is completely down-regulated as cells grow beyond about 50% confluency and no PKC-epsilon protein can be detected in 3T6 fibroblasts at high density by Western blotting. However, mRNA for PKC-epsilon is expressed at all stages of fibroblast growth as revealed by RT-PCR. When high-density 3T6 fibroblasts are passaged to low density in fresh medium, re-expression of PKC-epsilon protein is observed within 15 min and becomes down-regulated again as cells become more dense. This very rapid synthesis of PKC-epsilon is not blocked by the transcription inhibitor actinomycin D but is inhibited by cycloheximide. PKC-epsilon has some characteristics of a novel 'early response' protein whose synthesis in newly passaged 3T6 cells is regulated at the translational level.

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.

Reduced daunomycin accumulation in drug-sensitive and multidrug-resistant human carcinoma KB cells following phorbol ester treatment: a potential role for protein kinase C in reducing drug influx.

We have investigated the role of protein kinase C (PKC) in the multidrug resistance (MDR) phenotype of human KB carcinoma cell lines. The PKC activator 12-O-tetradecanoylphorbol-13-acetate (TPA) reduced daunomycin accumulation in both drug-sensitive KB-3-1 and MDR KB-C1 cells in a time-dependent manner. The inactive phorbol ester 4 alpha TPA did not reduce daunomycin accumulation, and the PKC inhibitor, Ro 31-8220, reversed the TPA effect. TPA had no effect on daunomycin efflux and did not induce Pgp expression in KB-3-1 cells or alter Pgp levels in KB-C1 cells. Linear, short-term daunomycin accumulation was reduced by pretreatment with TPA, an effect that could be reversed by Ro 31-8220. The effects of TPA on PKC subspecies localisation and downregulation were also examined. TPA initially induced translocation of PKCs alpha and delta, and to a lesser extent, PKC epsilon to the membrane fraction; 8 h after TPA treatment, differential effects on downregulation of PKCs alpha and delta were observed between cell lines, although PKC epsilon was not reduced in either cell line. We therefore propose that the TPA-induced reduction in daunomycin accumulation in KB cells is due to a PKC-mediated process, which is maintained after depletion of certain PKC subspecies or is due to activation of downregulation insensitive PKC subspecies. These results suggest that PKC may regulate drug resistance by reducing drug influx in a Pgp-independent manner in KB cells. This may represent a mechanism of drug-resistance independent of, or in addition, to, Pgp-mediated drug efflux.

Protein expression of the alpha, gamma, delta and epsilon subspecies of protein kinase C changes as C6 glioma cells become contact inhibited and quiescent in the presence of serum.

Total protein kinase C (PKC) activity and protein expression of the alpha and delta subspecies of PKC increases markedly as C6 glioma cells grow from low cell density to the contact-inhibited quiescent state (also known as G(o)) in the presence of serum. At the same time protein expression of PKC subspecies gamma and epsilon decreases while the beta I, beta II, iota and zeta subspecies did not change. Serum deprivation of growing C6 glioma cells does not induce the same changes in PKC subspecies protein expression. The findings support the growing view that there are significant differences between the G(o) states brought about by contact inhibition or serum deprivation.