Effects of mevinolin treatment on tissue dolichol and ubiquinone levels in the rat.

Rats were treated with mevinolin by intraperitoneal injection (15 days) or dietary administration (30 days). The cholesterol, dolichol, dolichyl phosphate and ubiquinone contents of the liver, brain, heart, muscle and blood were then investigated. The cholesterol contents of these organs did not change significantly, with the exception of muscle. Intraperitoneal administration of the drug increases the amount of dolichol in liver, muscle and blood and decreases the dolichyl-P amount in muscle. The same treatment increases the level of ubiquinone in muscle and blood and decreases this value in liver and heart. Oral administration decreases dolichol, dolichyl-P and ubiquinone levels in heart and muscle, while in liver the dolichol level is elevated and ubiquinone level lowered. In brain the amount of dolichyl-P is increased. Intraperitoneal injection of mevinolin also modifies the liver dolichol and dolichyl-P isoprenoid pattern, with an increase in shorter chain polyisoprenes. The levels of dolichol and ubiquinone in the blood do not follow the changes observed in other tissues. Incorporation of [3H]acetate into cholesterol by liver slices prepared from mevinolin-treated rats exhibited an increase, whereas in brain no change was seen. Labeling of dolichol and ubiquinone was increased in both liver and brain, but incorporation into dolichyl phosphate remained relatively stable. The results indicate that mevinolin affects not only HMG-CoA reductase but, to some extent, also affects certain of the peripheral enzymes, resulting in considerable effects on the various mevalonate pathway lipids.     

Relation of mevalonate synthesis to mitochondrial ubiquinone content and respiratory function in cultured neuroblastoma cells

The consequence of blocking the de novo synthesis of ubiquinone (coenzyme Q) on mitochondrial ubiquinone content and respiratory function was studied in cultured C1300 (Neuro 2A) murine neuroblastoma cells. Mevinolin, a competitive inhibitor of 3-hydroxy-3-methylglutaryl coenzyme A reductase, was used to suppress the synthesis of mevalonate, an essential precursor for the isoprenoid side chain of ubiquinone. At a concentration of 25 microM, mevinolin completely inhibited the incorporation of [3H]acetate into ubiquinone, isolated from cell extracts by two-dimensional thin-layer chromatography. Similar results were obtained when [14C]tyrosine was used as a precursor for the quinone ring. Through the use of reverse-phase thin-layer chromatography, it was established that the principal product of the ubiquinone pathway in murine neuroblastoma cells was ubiquinone-9. Inhibition of ubiquinone synthesis for 24h in cells cultured in the presence of 10% fetal calf serum (which contains 0.14 nmol of ubiquinone/ml of serum) resulted in a 40-57% decline in the concentration of ubiquinone in the mitochondria. However, the activities of succinate-cytochrome c reductase and succinate dehydrogenase in whole-cell homogenates or mitochondria were not inhibited. The state 3 and uncoupled rates of respiration, determined by polarographic measurements of oxygen consumption in homogenates and mitochondria, were elevated slightly in the mevinolin-treated cells. The data demonstrate that, although mevalonate synthesis is important for the maintenance of the intramitochondrial ubiquinone pool in cultured cells, major changes in the ubiquinone content of the mitochondria can occur in intact cells without perturbation of respiratory function. However, the coincidence of decreased mitochondrial ubiquinone concentration and the inhibition of cell cycling previously observed in mevinolin-treated cells (Maltese, W.A. (1984) Biochem. Biophys. Res. Commun. 120, 454-460) suggests that the availability of ubiquinone may play a role in the regulation of mitochondrial and cellular proliferation.     

Effects of prior sterol depletion on neurite outgrowth in neuroblastoma cells

The effect of decreasing cellular sterol content on neurite outgrowth in C1300 (Neuro 2A) neuroblastoma cells in serum-free medium has been studied. Sterol-depleted, undifferentiated neuroblastoma cells were obtained by growing cells for 24 h in medium containing lipoprotein-poor serum and 25-hydroxy-cholesterol (25-OHC). Under these conditions the activity of 3-hydroxy-3-methyl-glutaryl-CoA reductase and the incorporation of [₁₄C] acetate into sterols were almost completely suppressed, and the sterol/phospholipid ratio of the cells declined to 60% of that in cultures grown without 25-OHC. The sterol-depleted cells were viable and exhibited rates of DNA, RNA, protein and fatty acid synthesis comparable to those measured in control cultures. Sterol depletion had no detectable effect on the number of cells that were able to undergo morphological differentiation within 3 h after removal of serum from the medium. However, by 24 h most of the sterol-depleted cells had retracted their neurites. The observation that addition of low-density lipoprotein was able to restore neurite outgrowth in cultures treated with 25-OHC indicates that the inability of sterol-depleted cells to maintain their neurites is related specifically to the decline in the sterol content rather than to a general cytotoxic effect of 25-OHC. Our findings suggest that incorporation of cholesterol into the cell membrane is important for long-term maintenance and elongation of neuroblastoma neurites, but that the initial morphological change (i.e., within 3 h after removal of serum) is apparently a separate and distinct event, not dependent on the availability of cholesterol.     

Induction of differentiation in murine neuroblastoma cells by mevinolin, a competitive inhibitor of 3-hydroxy-3-methylglutaryl coenzyme A reductase

Mevinolin, a competitive inhibitor of 3-hydroxy-3-methylglutaryl coenzyme A reductase, stimulated outgrowth of neurites and increased acetylcholinesterase activity in C1300-N2A murine neuroblastoma cells cultured in medium containing 10% fetal calf serum. Changes in cell morphology and enzyme activity were concentration-dependent in the range of 0.25-25 microM mevinolin, and were accompanied by decreased incorporation of [3H]thymidine into DNA. The expression of differentiated characteristics induced by 25 microM mevinolin was blocked by simultaneous addition of 100 microM mevalonate to the culture medium. The data suggest that changes in intracellular levels of mevalonate or one of its isoprenoid derivatives may play a role in the regulation of cell differentiation.     

Effects of an inhibitor of glucosylceramide synthase on glycosphingolipid synthesis and neurite outgrowth in murine neuroblastoma cell lines.

1-Phenyl-2-decanolyamino-3-morpholino-1-propanol (PDMP), an effective inhibitor of UDP-glucose:ceramide glucosyltransferase, caused inhibition of cell growth in murine neuroblastoma cell lines. Metabolic labeling of glycosphingolipids with [14C]galactose in NS-20Y, Neuro2a, and N1E-115 cells showed reduced incorporation of radioactivity into gangliosides and neutral glycosphingolipids when threo-PDMP was present in the medium. Treatment of NS-20Y cells with threo-PDMP resulted in a time-dependent decrease in mass levels of gangliosides and neutral glycosphingolipids. After 24 h in the presence of 50 microM threo-PDMP, neutral glycosphingolipid mass was reduced to 32%, where glucosylceramide was the most affected (90% decrease). The ganglioside mass was reduced to 57% of the original content. Neurite outgrowth from neuroblastoma cells in serum-free medium was significantly inhibited by threo-PDMP in a dose-dependent manner. Threo-PDMP also caused retraction of neurites which had been induced to extend in serum-free medium. Pretreatment of cells with GM1 partially restored the ability of NS-20Y cells for neurite outgrowth in the medium containing threo-PDMP. These results suggest a possible role for glycosphingolipids in neurite outgrowth of murine neuroblastoma cells.     

Isoprenoids and astroglial cell cycling: diminished mevalonate availability and inhibition of dolichol-linked glycoprotein synthesis arrest cycling through distinct mechanisms.

Primary astroglial cultures were used to compare the relationships to cell cycling of dolichol-linked glycoprotein synthesis, and of availability of mevalonate, the precursor of dolichol and other isoprenoid lipids. With shift-up to 10% serum (time 0) after 48 h of serum depletion, the proportion of cells in S phase (bromodeoxyuridine immunofluorescence) remained under 15% for 12 h, then increased by 20 h to 72 +/- 10%; DNA synthetic rates (thymidine incorporation) increased 5-fold. S phase transition was prevented by addition at 10-12 h of tunicamycin, an inhibitor of transfer of saccharide moieties to dolichol. Mevinolin, an inhibitor of mevalonate biosynthesis, also blocked cycle progression when added at this time. However, mevinolin markedly inhibited the isoprenoid pathway, as reflected by over 90% reduction of sterol synthesis, without inhibiting net glycoprotein synthesis. Removal of mevinolin after a 24 h exposure delayed S phase until 48 h, following recovery of sterol synthesis, even though kinetics of glycoprotein synthesis were unaffected. Tunicamycin removal after 24 h spared sterol synthesis, but caused delay of S phase until 72 h, following recovery of glycoprotein synthesis. In mevinolin-treated cultures, S phase transition was restored by 1 h of exposure to mevalonate at 10 h, although cycling was thereby rendered sensitive to inhibition by cycloheximide and by tunicamycin. Cell cycle progression following hydroxyurea exposure and release was unaffected by mevinolin, tunicamycin, or cycloheximide. Thus, in these developing astroglia, mevalonate and its isoprenoid derivatives have at least two cell cycle-specific roles: dolichol-linked glycoprotein synthesis is required at or before the G1/S transition, while a distinct mevalonate requirement is apparent also in late G1.     

Neurite outgrowth activity of protease nexin-1 on neuroblastoma cells requires thrombin inhibition

Protease nexin-1 (PN-1) is a protein proteinase inhibitor recently shown to be identical with the glial-derived neurite-promoting factor or glial-derived nexin. It has been shown to promote neurite outgrowth in neuroblastoma cells and in sympathetic neurons. The present experiments were designed to further test the hypothesis that this activity on neuroblastoma cells is due to its ability to complex and inhibit thrombin. It has been suggested that PN-1:thrombin complexes might mediate the neurite outgrowth activity of PN-1. However, the present studies showed that such complexes, unlike free PN-1, did not promote neurite outgrowth. The neurite outgrowth activity of PN-1 was only detected in the presence of thrombin or serum (which contains thrombin). PN-1 did not affect the rate or extent of neurite outgrowth that occurred when neuroblastoma cells were placed in serum-free medium. Retraction of neurites by thrombin was indistinguishable in cells whose neurites had been extended in the presence or absence of PN-1. The neurite-promoting activity of PN-1 was inhibited by an anti-PN-1 monoclonal antibody, which blocks its capacity to complex serine proteinases. The plasma thrombin inhibitor, antithrombin III, stimulated neurite outgrowth but only when its thrombin inhibitory activity was accelerated by heparin. The neurite outgrowth activity of both antithrombin III and PN-1 corresponded to their inhibition of thrombin. Together, these observations show that PN-1 promotes neurite outgrowth from neuroblastoma cells by inhibiting thrombin and suggest that this depends on the ability of thrombin to retract neurites.     

Proteolytic regulation of neurite outgrowth from neuroblastoma cells by thrombin and protease nexin-1

This review summarizes studies on the reciprocal regulation of neuroblastoma neurite outgrowth by thrombin and protease nexin-1 (PN-1). PN-1 recently was shown to possess the same deduced amino acid sequence as the glial-derived neurite-promoting factor. The neurite outgrowth activity of PN-1 depends on its ability to inhibit thrombin. Thrombin not only blocks the neurite outgrowth activity of PN-1, but it also brings about neurite retraction in the presence of PN-1. Thrombin also produces neurite retraction in the absence of PN-1 and other regulatory factors. This suggests that its activity is due to a direct action on cells. The neurite retraction by thrombin depends on its proteolytic activity. It does not occur with the other serine proteases that have been tested, indicating that it is a specific effect and is not due to a general proteolytic effect that could detach neurites from the culture dish. Serum brings about neurite retraction in certain neuroblastoma cells and primary neuronal cultures; most of this activity is due to residual thrombin in the serum. Together, these results suggest that PN-1 and thrombin (or a thrombin-like protease) play a role in regulation of neurite outgrowth.     

Proteomics analysis of the expression of neurogranin in murine neuroblastoma (Neuro-2a) cells reveals its involvement for cell differentiation

Neurogranin (Ng) is a neural-specific, calmodulin (CaM)-binding protein that is phosphorylated by protein kinase C (PKC). Although its biochemical property has been well characterized, the physiological function of Ng needs to be elucidated. In the present study, we performed proteomics analysis of the induced compositional changes due to the expression of Ng in murine neuroblastoma (Neuro-2a) cells using isotope coded affinity tags (ICAT) combined with 2-dimensional liquid chromatography/tandem mass spectrometry (2D-LC/MS/MS). We found that 40% of identified proteins were down-regulated and most of these proteins are microtubule components and associated proteins that mediated neurite outgrowth. Western blot experiments confirmed the expression of alpha-tubulin and microtubule- associated protein 1B (MAP 1B) was dramatically reduced in Neuro-2a-Ng cells compared to control. Cell morphology of Neuro-2a-Ng showed far less neurites than the control. Serum deprivation induced the extension of only one or two long neurites per cell in Neuro-2a-Ng, contrasting to the extension of multiple neurites per control cell. Ng may be linked to neurite formation by affecting expression of several microtubule related proteins. Furthermore, the PKC activator (PMA) induced an enhanced ERK1/2 activity in the cells that expressed Ng. The mutation of Ng at S36A caused sustained increase of ERK1/2 activity, whereas the ERK1/2 activity in mutation at I33Q showed no difference compared to wild type Ng, suggesting the phosphorylation of Ng but not the CaM /Ng interaction plays an important role in ERK activation. Ng may be involved in neuronal growth and differentiation via PKC and ERK1/2 signaling pathways.     

Autonomous and non-autonomous regulation of mammalian neurite development by Notch1 and Delta1

BACKGROUND: On the basis of experiments suggesting that Notch and Delta have a role in axonal development in Drosophila neurons, we studied the ability of components of the Notch signaling pathway to modulate neurite formation in mammalian neuroblastoma cells in vitro. RESULTS: We observed that N2a neuroblastoma cells expressing an activated form of Notch, Notch1(IC), produced shorter neurites compared with controls, whereas N2a cell lines expressing a dominant-negative Notch1 or a dominant-negative Delta1 construct extended longer neurites with a greater number of primary neurites. We then compared the effects on neurites of contacting Delta1 on another cell and of overexpression of Delta1 in the neurite-extending cell itself. We found that N2a cells co-cultured with Delta1-expressing quail cells produced fewer and shorter neuritic processes. On the other hand, high levels of Delta1 expressed in the N2a cells themselves stimulated neurite extension, increased numbers of primary neurites and induced expression of Jagged1 and Notch1. CONCLUSIONS: These studies show that Notch signals can antagonize neurite outgrowth and that repressing endogenous Notch signals enhances neurite outgrowth in neuroblastoma cells. Notch signals therefore act as regulators of neuritic extension in neuroblastoma cells. The response of neuritic processes to Delta1 expressed in the neurite was opposite to that to Delta1 contacted on another cell, however. These results suggest a model in which developing neurons determine their extent of process outgrowth on the basis of the opposing influences on Notch signals of ligands contacted on another cell and ligands expressed in the same cell.     

Induction of neurites by the regulatory domains of PKCdelta and epsilon is counteracted by PKC catalytic activity and by the RhoA pathway

We have shown that protein kinase C (PKC) epsilon, independently of its kinase activity, via its regulatory domain (RD), induces neurites in neuroblastoma cells. This study was designed to evaluate whether the same effect is obtained in nonmalignant neural cells and to dissect mechanisms mediating the effect. Overexpression of PKCepsilon resulted in neurite induction in two immortalised neural cell lines (HiB5 and RN33B). Phorbol ester potentiated neurite outgrowth from PKCepsilon-overexpressing cells and led to neurite induction in cells overexpressing PKCdelta. The effects were potentiated by blocking the PKC catalytic activity with GF109203X. Furthermore, kinase-inactive PKCdelta induced more neurites than the wild-type isoform. The isolated regulatory domains of novel PKC isoforms also induced neurites. Experiments with PKCdelta-overexpressing HiB5 cells demonstrated that phorbol ester, even in the presence of a PKC inhibitor, led to a decrease in stress fibres, indicating an inactivation of RhoA. Active RhoA blocked PKC-induced neurite outgrowth, and inhibition of the RhoA effector ROCK led to neurite outgrowth. This demonstrates that neurite induction by the regulatory domain of PKCdelta can be counteracted by PKCdelta kinase activity, that PKC-induced neurite outgrowth is accompanied by stress fibre dismantling indicating an inactivation of RhoA, and that the RhoA pathway suppresses PKC-mediated neurite outgrowth.     

Myosin IIA drives neurite retraction

Neuritic extension is the resultant of two vectorial processes: outgrowth and retraction. Whereas myosin IIB is required for neurite outgrowth, retraction is driven by a motor whose identity has remained unknown until now. Preformed neurites in mouse Neuro-2A neuroblastoma cells undergo immediate retraction when exposed to isoform-specific antisense oligonucleotides that suppress myosin IIB expression, ruling out myosin IIB as the retraction motor. When cells were preincubated with antisense oligonucleotides targeting myosin IIA, simultaneous or subsequent addition of myosin IIB antisense oligonucleotides did not elicit neurite retraction, both outgrowth and retraction being curtailed. Even during simultaneous application of antisense oligonucleotides against both myosin isoforms, lamellipodial spreading continued despite the complete inhibition of neurite extension, indicating an uncoupling of lamellipodial dynamics from movement of the neurite. Significantly, lysophosphatidate- or thrombin-induced neurite retraction was blocked not only by the Rho-kinase inhibitor Y27632 but also by antisense oligonucleotides targeting myosin IIA. Control oligonucleotides or antisense oligonucleotides targeting myosin IIB had no effect. In contrast, Y27632 did not inhibit outgrowth, a myosin IIB-dependent process. We conclude that the conventional myosin motor, myosin IIA, drives neurite retraction.     

XIB4035, a novel nonpeptidyl small molecule agonist for GFRalpha-1

We investigated the agonistic activities of N(4)-(7-chloro-2-[(E)-2-(2-chloro-phenyl)-vinyl]-quinolin-4-yl)-N(1),N(1)-diethyl-pentane-1,4-diamine (XIB4035), at the glial cell line-derived neurotrophic factor (GDNF) family receptoralpha-1(GFRalpha-1) in Neuro-2A cells, a mouse neuroblastoma cell line which is a suitable model for investigating functions mediated through GFRalpha-1. XIB4035 concentration-dependently inhibited [(125)I]GDNF binding in Neuro-2A cells with an IC(50) of 10.4 microM. GDNF induced autophosphorylation of Ret protein, and promoted neurite outgrowth in Neuro-2A cells. XIB4035, like GDNF, induced Ret autophosphorylation in the Neuro-2A cells. Moreover, XIB4035 promoted neurite outgrowth in a concentration-dependent manner. These results show that XIB4035 may act as an agonist at GFRalpha-1 receptor complex, and mimic neurotrophic effects of GDNF in Neuro-2A cells. This is an interesting finding showing that a nonpeptidyl small molecule is capable of inducing activation of a receptor that normally bind a relatively large protein ligand such as GDNF.     

Cellular cholesterol metabolism in mitogen-stimulated lymphocytes--requirement for de novo synthesis

The relationship between cholesterol synthesis and uptake in proliferating lymphocytes has been examined. [14C]Acetate incorporation into lymphocytes cultured under lipoprotein-deficient conditions increased initially in response to mitogen, decreased after 24 h, and increased rapidly between 72 and 96 h. Addition of LDL (10 micrograms/ml) to the culture during the 'trough' period caused [14C]acetate incorporation to return rapidly to baseline, while at peak periods LDL suppression of cholesterol synthesis was minimal. Lymphocytes cultured in the presence of the HMG-CoA reductase inhibitor, mevinolin, exhibited a time-dependent increase in their capacity to incorporate [14C]acetate into cholesterol, evident when mevinolin was removed by washing prior to assay. PHA enhanced 125I-labelled LDL receptor-mediated binding by lymphocytes cultured in lipoprotein-deficient medium over a 4 day period and mevinolin augmented the effect. [3H]Thymidine incorporation into mitogen-stimulated lipoprotein-deficient cultures was inhibited up to 75% by mevinolin (1 mumol/l). LDL (2.5-10 micrograms/ml) substantially reversed this inhibition in 72 h cultures, but only partially overcame inhibition in cells cultured for 96 h. Results suggest that endogenous cholesterol synthesis may be obligatory for lymphocyte proliferation after the initial round of cell division.     

Phosphatidylinositol 3-kinase, Cdc42, and Rac1 act downstream of Ras in integrin-dependent neurite outgrowth in N1E-115 neuroblastoma cells

Ras and Rho family GTPases have been ascribed important roles in signalling pathways determining cellular morphology and growth. Here we investigated the roles of the GTPases Ras, Cdc42, Rac1, and Rho and that of phosphatidylinositol 3-kinase (PI 3-kinase) in the pathway leading from serum starvation to neurite outgrowth in N1E-115 neuroblastoma cells. Serum-starved cells grown on a laminin matrix exhibited integrin-dependent neurite outgrowth. Expression of dominant negative mutants of Ras, PI 3-kinase, Cdc42, or Rac1 all blocked this neurite outgrowth, while constitutively activated mutants of Ras, PI 3-kinase, or Cdc42 were each sufficient to promote outgrowth even in the presence of serum. A Ras(H40C;G12V) double mutant which binds preferentially to PI 3-kinase also promoted neurite formation. Activated Ras(G12V)-induced outgrowth required PI 3-kinase activity, but activated PI 3-kinase-induced outgrowth did not require Ras activity. Although activated Rac1 by itself did not induce neurites, neurite outgrowth induced by activated Cdc42(G12V) was Rac1 dependent. Cdc42(G12V)-induced neurites appeared to lose their normal polarization, almost doubling the average number of neurites produced by a single cell. Outgrowth induced by activated Ras or PI 3-kinase required both Cdc42 and Rac1 activity, but Cdc42(G12V)-induced outgrowth did not need Ras or PI 3-kinase activity. Active Rho(G14V) reduced outgrowth promoted by Ras(G12V). Finally, expression of dominant negative Jun N-terminal kinase or extracellular signal-regulated kinase did not inhibit outgrowth, suggesting these pathways are not essential for this process. Our results suggest a hierarchy of signalling where Ras signals through PI 3-kinase to Cdc42 and Rac1 activation (and Rho inactivation), culminating in neurite outgrowth. Thus, in the absence of serum factors, Ras may initiate cell cycle arrest and terminal differentiation in N1E-115 neuroblastoma cells.     

Endogenous GM1 Ganglioside of the Plasma Membrane Promotes Neuritogenesis by Two Mechanisms

The influence of GM1 on the neuritogenic phase of neuronal differentiation has been highlighted in recent reports showing upregulation of this ganglioside in the plasma and nuclear membranes concomitant with axonogenesis. These changes are accompanied by alterations in Ca²⁺ flux which constitute an essential component of the signaling mechanism for axon outgrowth. This study examines 2 distinct mechanisms of induced neurite outgrowth involving plasma membrane GM1, as expressed in 3 neuroblastoma cell lines. Growth of Neuro-2a and NG108-15 cells in the presence of neuraminidase (N'ase), an enzyme that increases the cell surface content of GM1, caused prolific outgrowth of neurites which, in the case of Neuro-2a, could be blocked by the B subunit of cholera toxin (Ctx B) which binds specifically to GM1; however, the latter agent applied to NG108-15 cells proved neuritogenic and potentiated the effect of N'ase. With N18 cells, the combination was also neuritogenic as was Ctx B alone, whereas N'ase by itself had no effect. Neurite outgrowth correlated with influx of extracellular Ca²⁺, determined with fura-2. Treatment of NG108-15 and N18 cells with Ctx B alone caused modest but persistent elevation of intracellular Ca²⁺ while a more pronounced increase occurred with the combination Ctx B + N'ase. Treatment with N'ase alone also caused modest but prolonged elevation of intracellular Ca²⁺ in NG108-15 and Neuro-2a but not N18; in the case of Neuro-2a this effect was blocked by Ctx B. Neuro-2a and N18 thus possess 2 distinctly different mechanisms for neuritogenesis based on Ca²⁺ modulation by plasma membrane GM1, while NG108-15 cells show both capabilities. The neurites stimulated by N'ase + Ctx B treatment of N18 cells were shown to have axonal character, as previously demonstrated for NG108-15 cells stimulated in this manner and for Neuro-2a cells stimulated by N'ase alone.     

Isoprenylated proteins in cultured cells: subcellular distribution and changes related to altered morphology and growth arrest induced by mevalonate deprivation

In the presence of lovastatin (mevinolin), an inhibitor of endogenous mevalonate synthesis, C1300 murine neuroblastoma cells incorporated (2-14C)mevalonate into several discrete polypeptides that were separable by SDS-PAGE. The electrophoretic pattern of the labeled proteins did not vary substantially when cells were homogenized with Ca++, Mg++, high concentrations of NaCl or phosphatase inhibitor, or when cells were lysed immediately in trichloroacetic acid. When cells that had been prelabeled with (14C)mevalonate were incubated with lovastatin and simultaneously deprived of exogenous mevalonate, there was a 50-60% decline in the concentration of protein-bound isoprenoid label within 17 h. In contrast, there was little change in the radioactivity in the sterol, dolichol, or ubiquinone fractions. The time course of the decline in mevalonate-derived label in cellular polypeptides paralleled the onset of neurite outgrowth and preceded the decline of DNA synthesis, suggesting that a decreased intracellular concentration of protein-bound isoprenoid groups may contribute to the well-documented effects of mevalonate deprivation on cell morphology and cell cycling. Fractionation of neuroblastoma cells by differential centrifugation and sucrose density-gradient centrifugation revealed that mevalonate-labeled proteins of 53 kDA, 22-26 kDa, and 17 kDa were concentrated in the cytosol. Proteins migrating at 45 kDa were found in both the soluble and particulate fractions, including those enriched in mitochondria and plasma membrane. The isoprenylated proteins migrating at approximately 66 kDa were localized exclusively in the nuclear fraction. When chromatin was removed from the nuclei by extraction with 2 M NaCl, the 66 kDa isoprenylated proteins remained associated with the residual components of the nuclear matrix and lamina. Isoprenylated proteins with electrophoretic mobilities similar to those observed in neuroblastoma cells were detected in a variety of established cell lines. However, there was considerable variation among cell lines in the overall efficiency of protein labeling with (14C) mevalonate and in the prominence and mobilities of specific labeled proteins in the 45-70 kDa range. Comparisons of paired transformed vs. nontransformed fibroblast cell lines suggested that the profile of mevalonate-labeled proteins in a given cell line is not altered by malignant transformation.(ABSTRACT TRUNCATED AT 400 WORDS)     

Stimulation of anchorage independent proliferation of human adrenocortical carcinoma cells by inhibition of cholesterol biosynthesis

Proliferation of SW13 human adrenocortical carcinoma cells under anchorage independent conditions was stimulated in a dose-dependent manner by treatment with the cholesterol biosynthesis inhibitor mevinolin. Induction of 3-hydroxy-3-methylglutaryl coenzyme A reductase activity was observed in mevinolin treated cultures. The growth stimulatory effect of mevinolin, but not that of epithelial transforming growth factor, a polypeptide growth factor for SW13 cells, was reversed by exogenous mevalonic acid. However, neither dolichol nor low density lipoprotein supplementation affected the response of SW13 cells to mevinolin. The results suggest that mevalonic acid metabolites may participate in the regulation of anchorage independent growth of SW13 cells.     

Effects of apolipoprotein E, beta-very low density lipoproteins, and cholesterol on the extension of neurites by rabbit dorsal root ganglion neurons in vitro.

Previous studies suggest that during nerve regeneration apoE acts as a lipid transport protein that assists in the rapid initial extension of axons and then in their myelination. To determine whether apoE and/or apoE-containing lipoproteins can modulate axon growth, we assessed their effect on the out-growth of neurites from neurons in mixed cultures of fetal rabbit dorsal root ganglion cells in vitro. Incubation with beta-very low density lipoprotein (beta-VLDL) particles, which are rich in apoE and cholesterol, increased neurite outgrowth and branching. Unesterified cholesterol added to the cultures had a similar, but less pronounced, effect. These data suggest that cholesterol might be the component responsible for the enhanced neurite growth. In contrast, purified, lipid-free apoE added to the cultures reduced neurite branching. Neurite branching was also reduced when purified apoE was added along with beta-VLDL or cholesterol; however, the striking finding was that under these conditions the neurites extended farther from the neuronal cell body. Dorsal root ganglion cells were examined for the presence of receptors for native and apoE-enriched beta-VLDL. Immunocytochemistry, ligand blots, 45Ca2+ blots, and studies of the interaction of the cells with fluorescent lipoproteins provided evidence of two types of receptors for apoE-containing lipoproteins on neurons: the low density lipoprotein (LDL) receptor, which binds native beta-VLDL, and the LDL receptor-related protein, which binds apoE-enriched beta-VLDL. These findings indicate that apoE may play two complementary roles in neurite outgrowth. When complexed with lipoproteins, apoE stimulates neurite growth by the receptor-mediated delivery of cholesterol and perhaps other components necessary for neurite outgrowth. When apoE as a free protein is added together with apoE-containing lipoproteins, apoE decreases neurite branching and promotes neurite extension away from the cell body. These actions, which would be complementary in promoting target-directed nerve growth in vivo, provide the first direct evidence that apoE and apoE-containing lipoproteins can modulate the outgrowth of neuronal processes.     

Multiple Neurite Formation in Neuroblastoma Cell Lines by Griseolic Acid, a Potent Inhibitor of Cyclic Nucleotide Phosphodiesterases

The morphological change of several neuroblastoma cell lines induced by griseolic acid, a novel and potent inhibitor of cyclic nucleotide phosphodiesterase (PDE), was examined. In the cell lines tested, Neuro-2a (a murine neuroblastoma cell line) showed dose-dependent (1 microM-1 mM) neurite extension. Griseolic acid markedly increased the intracellular cyclic AMP level of Neuro-2a cells, suppressed DNA synthesis (82% at 1 mM), and induced multipolar (multiple-neurite-bearing)-type neuritogenesis. A similar type of neurite outgrowth was induced by 8-bromo-cyclic AMP, which also elevated the intracellular cyclic AMP concentration. In contrast, when Neuro-2a cells were treated with retinoic acid, neurite formation was of the monopolar (single-neurite-bearing) type. Papaverine and theophylline, which have been frequently used as PDE inhibitors, failed to induce these morphological changes up to 1 mM, probably owing to the lesser potency of these compounds as compared with griseolic acid on the inhibition of PDE. Retinoic acid, theophylline, and papaverine were ineffective at elevating the intracellular cyclic AMP level. These results suggest that multipolar-type cell shape change in Neuro-2a cells is correlated with the accumulation of intracellular cyclic AMP and that griseolic acid is a useful compound to induce neuroblastoma cells into terminal differentiation.