Synthetic analogs of FTY720 [2-amino-2-(2-[4-octylphenyl]ethyl)-1,3-propanediol] differentially regulate pulmonary vascular permeability in vivo and in vitro.

Novel therapies are needed to address the vascular endothelial cell (EC) barrier disruption that occurs in inflammatory diseases such as acute lung injury (ALI). We previously demonstrated the potent barrier-enhancing effects of both sphingosine 1-phosphate (S1P) and the structurally similar compound FTY720 [2-amino-2-(2-[4-octylphenyl]ethyl)-1,3-propanediol] in inflammatory lung injury. In this study, we examined the therapeutic potential of several novel FTY720 analogs to reduce vascular leak. Similar to S1P and FTY720, the (R)- and (S)-enantiomers of FTY720 phosphonate and enephosphonate analogs produce sustained EC barrier enhancement in vitro, as seen by increases in transendothelial electrical resistance (TER). In contrast, the (R)- and (S)-enantiomers of FTY720-regioisomeric analogs disrupt EC barrier integrity in a dose-dependent manner. Barrier-enhancing FTY720 analogs demonstrate a wider protective concentration range in vitro (1-50 microM) and greater potency than either S1P or FTY720. In contrast to FTY720-induced EC barrier enhancement, S1P and the FTY720 analogs dramatically increase TER within minutes in association with cortical actin ring formation. Unlike S1P, these FTY720 analogs exhibit differential phosphorylation effects without altering the intracellular calcium level. Inhibitor studies indicate that barrier enhancement by these analogs involves signaling via G(i)-coupled receptors, tyrosine kinases, and lipid rafts. Consistent with these in vitro responses, the (S)-phosphonate analog of FTY720 significantly reduces multiple indices of alveolar and vascular permeability in a lipopolysaccharide-mediated murine model of ALI (without significant alterations in leukocyte counts). These results demonstrate the capacity for FTY720 analogs to significantly decrease pulmonary vascular leakage and inflammation in vitro and in vivo.

Novel ceramide analogues display selective cytotoxicity in drug-resistant breast tumor cell lines compared to normal breast epithelial cells.

The sphingolipid ceramide is involved in diverse cell signaling pathways related to proliferation and differentiation. Elevated ceramide also triggers apoptosis. Synthetic ceramide derivatives have been shown to be cytotoxic to tumors, yet few studies have evaluated whether cytotoxicity of synthetic ceramides is selective for tumor cells. We have evaluated the cytotoxic potency of several novel ceramide analogues in the drug-resistant breast tumor cell lines, SKBr3 and MCF-7/Adr, and compared their cytotoxicity in normal breast epithelial cells. Cytotoxicity was assessed using release of lactate dehydrogenase into the culture medium. (2S, 3S)-3-(6'-Dodecylpyridin-2'-yl)-2-butanoylamidopropane-1,3-diol (pyridine-C4-ceramide) produced non-selective cytotoxicity across the three cell types (EC50= 12.8-16.7 microM, at 24 hr). However, 2S,5R-2-(octanoylamido-(3E))-octadecene-1,5-diol (5R-OH-3E-C8-ceramide), (2S,3R)-2-(N-adamantoyl)-(4E)-octadecen-1,3-diol (adamantyl-ceramide), and (2S,3R)-3-(3'-dodecylphenyl)-2-butanoylamidopropane-1,3-diol (benzene-C4-ceramide) exhibited increased cytotoxicity in the tumor cell lines compared to the normal breast epithelial cells. The EC50 values (microM) at 24 hr for these compounds in SKBr3 cells, MCF-7/Adr cells, and normal breast epithelial cells, respectively, were as follows: 5R-OH-3E-C8-ceramide, 18.3, 21.2 and 58.7; adamantyl-ceramide, 10.9, 24.9 and >100; benzene-C4-ceramide, 18.9, 45.5 and >100. At a concentration of 30 microM, the fold increase in cytotoxicity in breast tumor cell lines compared with normal breast epithelial cells was as follows: 5R-OH-3E-C8-ceramide, 23.7 and 19; adamantyl-ceramide, 11.2 and 10.3 and benzene-C4-ceramide, 79.3 and 77.2, for SKBr3 and MCF-7/Adr cells, respectively. Possible mechanisms accounting for selectivity are discussed. Ceramide analogues with relatively selective toxicity against tumor cells may have potential as therapeutic agents. Elucidating the mechanisms of selective cytotoxicity could identify novel targets that may lead to development of anti-neoplastic agents with a higher therapeutic index.

PE2 cleavage mutants of Sindbis virus: correlation between viral infectivity and pH-dependent membrane fusion activation of the spike heterodimer.

The spike glycoprotein E2 of Sindbis virus (SIN) is synthesized in the infected cell as a PE2 precursor protein, which matures through cleavage by a cellular furin-like protease. Previous work has shown that SIN mutants impaired in PE2 cleavage are noninfectious on BHK-21 cells, the block in infection being localized at a step after virus-receptor interaction but prior to RNA replication. Here, we studied the membrane fusion properties of SIN PE2 cleavage mutants and observed that these viruses are impaired in their ability to form an E1 homotrimer and to fuse with liposomes at a mildly acidic pH. The block in spike rearrangement and fusion could be overridden by exposure of the mutant viruses to very low pH (<4.5). Cleavage mutants with second-site resuscitating mutations in PE2 were highly infectious for BHK-21 cells. The ability of these viruses to form E1 homotrimers and to fuse at a mildly acidic pH was completely restored despite a sustained lack of PE2 cleavage.

Effect of the structure of natural sterols and sphingolipids on the formation of ordered sphingolipid/sterol domains (rafts). Comparison of cholesterol to plant, fungal, and disease-associated sterols and comparison of sphingomyelin, cerebrosides, and ceramide.

Ordered lipid domains enriched in sphingolipids and cholesterol (lipid rafts) have been implicated in numerous functions in biological membranes. We recently found that lipid domain/raft formation is dependent on the sterol component having a structure that allows tight packing with lipids having saturated acyl chains (Xu, X., and London, E. (2000) Biochemistry 39, 844-849). In this study, the domain-promoting activities of various natural sterols were compared with that of cholesterol using both fluorescence quenching and detergent insolubility methods. Using model membranes, it was shown that, like cholesterol, both plant and fungal sterols promote the formation of tightly packed, ordered lipid domains by lipids with saturated acyl chains. Surprisingly ergosterol, a fungal sterol, and 7-dehydrocholesterol, a sterol present in elevated levels in Smith-Lemli-Opitz syndrome, were both significantly more strongly domain-promoting than cholesterol. Domain formation was also affected by the structure of the sphingolipid (or that of an equivalent "saturated" phospholipid) component. Sterols had pronounced effects on domain formation by sphingomyelin and dipalmitoylphosphatidylcholine but only a weak influence on the ability of cerebrosides to form domains. Strikingly it was found that a small amount of ceramide (3 mol %) significantly stabilized domain/raft formation. The molecular basis for, and the implications of, the effects of different sterols and sphingolipids (especially ceramide) on the behavior and biological function of rafts are discussed.

Synthesis and growth inhibitory properties of glucosamine-derived glycerolipids.

[figure: see text] 2-Amino C-glycerolipid 1b was synthesized by using the Ramberg-Bäcklund rearrangement as the key step. beta-C-Glycerolipid 1b exhibits in vitro antiproliferative effects strikingly similar to those of O-glycoside analogue 1a.

Deacylation of the transmembrane domains of Sindbis virus envelope glycoproteins E1 and E2 does not affect low-pH-induced viral membrane fusion activity.

The envelope glycoproteins E1 and E2 of Sindbis virus are palmitoylated at cysteine residues within their transmembrane domains (E1 at position 430, and E2 at positions 388 and 390). Here, we investigated the in vitro membrane fusion activity of Sindbis virus variants (derived from the Toto 1101 infectious clone), in which the E1 C430 and/or E2 C388/390 residues had been substituted for alanines. Both the E1 and E2 mutant viruses, as well as a triple mutant virus, fused with liposomes in a strictly low-pH-dependent manner, the fusion characteristics being indistinguishable from those of the parent Toto 1101 virus. These results demonstrate that acylation of the transmembrane domain of Sindbis virus E1 and E2 is not required for expression of viral membrane fusion activity.

Coupling of cholesterol and cone-shaped lipids in bilayers augments membrane permeabilization by the cholesterol-specific toxins streptolysin O and Vibrio cholerae cytolysin.

Vibrio cholerae cytolysin (VCC) forms oligomeric pores in lipid bilayers containing cholesterol. Membrane permeabilization is inefficient if the sterol is embedded within bilayers prepared from phosphatidylcholine only but is greatly enhanced if the target membrane also contains ceramide. Although the enhancement of VCC action is stereospecific with respect to cholesterol, we show here that no such specificity applies to the two stereocenters in ceramide; all four stereoisomers of ceramide enhanced VCC activity in cholesterol-containing bilayers. A wide variety of ceramide analogs were as effective as D-erythro-ceramide, as was diacylglycerol, suggesting that the effect of ceramide exemplifies a general trend of lipids with a small headgroup to augment the activity of VCC. Incorporation of these cone-shaped lipids into cholesterol-containing bilayers also gave similar effects with streptolysin O, another cholesterol-specific but structurally unrelated cytolysin. In contrast, the activity of staphylococcal alpha-hemolysin, which does not share with the other toxins the requirement for cholesterol, was far less affected by the presence of lipids with a conical shape. The collective data indicate that sphingolipids and glycerolipids do not interact with the cytolysins specifically. Instead, lipids that have a conical molecular shape appear to effect a change in the energetic state of membrane cholesterol that in turn augments the interaction of the sterol with the cholesterol-specific cytolysins.

Methanolysis of sphingomyelin. Toward an epimerization-free methodology for the preparation of D-erythro-sphingosylphosphocholine.

It is well known that acid hydrolysis of natural sphingomyelin in aqueous methanol or 1-butanol at refluxing temperature is accompanied by epimerization at the C-3 position of the long-chain base. An improved procedure for the hydrolysis of commercially available, naturally occurring sphingomyelin is described. Prolonged exposure (3;-4 days) of sphingomyelin to freshly prepared 0.5 M anhydrous methanolic hydrogen chloride (generated by trapping the gas evolved from the reaction of concentrated sulfuric acid with solid sodium chloride in anhydrous methanol) at 50 degrees C resulted in cleavage of the amide side chain. The extent of epimerization of the allylic alcohol stereocenter was quantified by integration of the C-5 signal of the (13)C nuclear magnetic resonance spectrum of lysosphingomyelin. The method described here is superior to the traditional acid hydrolysis methods because it provides the product as a approximately 10:1 ratio of d-erythro/l-threo epimers; in contrast, a ratio of approximately 1. 3:1 was obtained by the previous methods. We also report that use of dichloromethane as a cosolvent with N,N-dimethylformamide in the reaction of lysosphingomyelin with an activated fatty acid reduced the time required for completion of the N-acylation reaction.

A stereocontrolled, efficient synthetic route to bioactive sphingolipids: synthesis of phytosphingosine and phytoceramides from unsaturated ester precursors via cyclic sulfate intermediates.

An efficient and highly enantioselective method for the preparation of D-ribo- and L-lyxo-phytosphingosines (1a,b, respectively) and phytoceramides (2a,b) has been developed. The key steps in the syntheses are as follows: (i) osmium-catalyzed asymmetric dihydroxylation of 4-O-protected (E)-alpha,beta-unsaturated ester 5 (generated by dihydroxylation of 1-hexadecene, followed by oxidation to the aldehyde and Horner-Wadsworth-Emmons olefination), (ii) conversion to cyclic sulfate intermediate 7, and (iii) regioselective alpha-azidation of 7. Reduction of 4-O-protected 2-azido ester 8 via alpha-azidolactone 9 afforded phytosphingosine 1a. Staudinger reduction of the azido group of 8, followed by in situ N-acylation in aqueous media and reduction of the ester functionality with NaBH(4)/LiBr, provided phytoceramide 2a. By using a similar approach, phytosphingosine 1b was synthesized. D-erythro-4, 5-Dihydrosphingosine 1c and D-erythro-4,5-dihydroceramide 2c were synthesized in high yield from 1-hexadecanol via cyclic sulfate intermediate 15. The desired configurations at C-2, C-3, and C-4 of the sphingoid chain can be accessed readily by the route described here.

Stereoselective preparation of ceramide and its skeleton backbone modified analogues via cyclic thionocarbonate intermediates derived by catalytic asymmetric dihydroxylation of alpha,beta-unsaturated ester precursors.

A novel and efficient synthetic route to ceramide 1a and skeleton backbone modified ceramide analogues 1b,c is reported. The syntheses utilize osmium-catalyzed asymmetric dihydroxylation of (E)-alpha, beta-unsaturated ester 5a-c as the chiral induction step, with the desired configurations in the products 1a-c, 2a, and 13 being generated by regioselective azide substitution at the alpha position of alpha,beta-dihydroxyesters 6a-c via a cyclic thionocarbonate intermediate. Azido esters 10a-c are converted to the corresponding ceramides 1a-c by a sequence of azide reduction, N-acylation, ester reduction (NaBH(4)/LiBr), and Birch reduction of the triple bond (Li, EtNH(2)). These seven- to eight-step syntheses afford the target compounds 1a-c with excellent stereocontrol and in 30-42% overall yields. Furthermore, propargylic alpha-azido-beta-hydroxyester 10a is converted to D-erythro-sphingosine 2a via simultaneous reduction of the triple bond, azido, and ester functional groups with LiAlH(4), providing a highly concise and practical four-step synthesis of this key naturally occurring sphingolipid. The L-erythro stereoisomers are also available in high enantiomeric purity by the method described herein.

Synthesis of ceramide analogues having the C(4)-C(5) bond of the long-chain base as part of an aromatic or heteroaromatic system.

Two efficient and stereoselective methods are described for the preparation of aryl and heteroaryl ceramide analogues 2 and 3. The first route involves the addition of an aryllithium or a heteroaryllithium reagent (7a or 25a, respectively) to the L-serine-derived aldehyde 4, followed by hydrolysis of the oxazolidine, liberation of the amino group, and N-acylation. The second route, which was used to prepare arylceramide analogue 2 in eight steps and 28% overall yield starting with 3-bromobenzaldehyde, utilizes a Heck reaction to afford (E)-alpha,beta-unsaturated ester 16, then osmium-catalyzed asymmetric dihydroxylation for the introduction of the desired chirality at C-2 and C-3. Regioselective alpha-azidation of alpha-O-nosyl-beta-hydroxyester 18 with sodium azide, followed by LiAlH(4) reduction of the azido and ester groups and N-acylation, complete the synthesis of arylceramide analogue 2.

Inhibition of protein kinase C(alpha) by dequalinium analogues: dependence on linker length and geometry.

Analogues of a bipartite compound, dequalinium (DECA) (quinolinium, 1,1'-(1,10-decanediyl)bis(4-amino-2-methyl diiodide)), were tested for inhibition of protein kinase C(alpha) (PKC(alpha)). In vitro assays of monomeric and dimeric analogues support a model in which DECA inhibits PKC(alpha) by an obligatory two-point contact, a unique mechanism among PKC inhibitors. The presence of unsaturation in the center of the C(10)-alkyl linker produced geometric isomers with different inhibitory potencies: cis IC(50) = 52 +/- 12 microM and trans IC(50) = 12 +/- 3 microM, where the trans isomer was equipotent to that of the saturated C(10)-DECA. DECA analogues with longer, saturated linkers (C(12), C(14), or C(16)) exhibited enhanced inhibitory potencies which reached a plateau with the C(14)-linker (IC(50) = 2.6 +/- 0.2 microM). Metastatic melanoma cells treated with 250 nM C(12)-, C(14)-, or C(16)-DECA and irradiated with long-wave UV light (which causes irreversible inhibition of PKC(alpha) by DECA) confirmed the linker-dependent inhibition of intracellular PKC(alpha) activity.

Low-pH-dependent fusion of Sindbis virus with receptor-free cholesterol- and sphingolipid-containing liposomes.

There is controversy as to whether the cell entry mechanism of Sindbis virus (SIN) involves direct fusion of the viral envelope with the plasma membrane at neutral pH or uptake by receptor-mediated endocytosis and subsequent low-pH-induced fusion from within acidic endosomes. Here, we studied the membrane fusion activity of SIN in a liposomal model system. Fusion was followed fluorometrically by monitoring the dilution of pyrene-labeled lipids from biosynthetically labeled virus into unlabeled liposomes or from labeled liposomes into unlabeled virus. Fusion was also assessed on the basis of degradation of the viral core protein by trypsin encapsulated in the liposomes. SIN fused efficiently with receptor-free liposomes, consisting of phospholipids and cholesterol, indicating that receptor interaction is not a mechanistic requirement for fusion of the virus. Fusion was optimal at pH 5.0, with a threshold at pH 6.0, and undetectable at neutral pH, supporting a cell entry mechanism of SIN involving fusion from within acidic endosomes. Under optimal conditions, 60 to 85% of the virus fused, depending on the assay used, corresponding to all of the virus bound to the liposomes as assessed in a direct binding assay. Preincubation of the virus alone at pH 5.0 resulted in a rapid loss of fusion capacity. Fusion of SIN required the presence of both cholesterol and sphingolipid in the target liposomes, cholesterol being primarily involved in low-pH-induced virus-liposome binding and the sphingolipid catalyzing the fusion process itself. Under low-pH conditions, the E2/E1 heterodimeric envelope glycoprotein of the virus dissociated, with formation of a trypsin-resistant E1 homotrimer, which kinetically preceded the fusion reaction, thus suggesting that the E1 trimer represents the fusion-active conformation of the viral spike.

Comparison of the capacity of beta-cyclodextrin derivatives and cyclophanes to shuttle cholesterol between cells and serum lipoproteins.

Previous studies from this laboratory have demonstrated that low concentrations of cyclodextrins (<1.0 mm), when added to serum, act catalytically as cholesterol shuttles to accelerate the exchange of free cholesterol between cells and serum lipoproteins. As cholesterol shuttles, cyclodextrins have the potential to serve as pharmacological agents for modifying cholesterol metabolism. In the present study, we have quantitated the cholesterol-shuttling capacity of a series of newly synthesized beta-cyclodextrin derivatives (betaCDs), with varying structure, and two double-decker cyclophanes. The general protocol is as follows. [(3)H]cholesterol-labeled CHOK1 cells are incubated for 2 h with the test compounds alone or together with 5% human serum, and efflux of the cellular [(3)H]cholesterol is measured. As methyl beta-cyclodextrin (MbetaCD) served as the basis for comparison, initial experiments were conducted that demonstrated there was a dose-dependent stimulation of cell cholesterol efflux as the concentration of MbetaCD increased, with an EC(50) that was calculated to be 0.05 mm. To determine the cholesterol-shuttling capacity of the newly synthesized compounds, cell cholesterol efflux is measured when the compounds are present alone, at a concentration of 0.05 mm, or together with 5% human serum. Our results demonstrate that the double-decker cyclophanes are the most efficient cholesterol shuttles. Under our experimental conditions, methyl beta-cyclodextrin (MbetaCD) approximately doubles the efflux of cell cholesterol to serum, whereas one of the double-decker cyclophanes produces a 4-fold stimulation in efflux. Four of the beta-cyclodextrin derivatives (betaCDs) display shuttling ability similar to that of MbetaCD. Furthermore, there does not appear to be a structural pattern among the other betaCDs which could explain their shuttling capacity.

Lysophosphatidic acid mediates the rapid activation of platelets and endothelial cells by mildly oxidized low density lipoprotein and accumulates in human atherosclerotic lesions.

Oxidized low density lipoprotein (LDL) is a key factor in the pathogenesis of atherosclerosis and its thrombotic complications, such as stroke and myocardial infarction. It activates endothelial cells and platelets through mechanisms that are largely unknown. Here, we show that lysophosphatidic acid (LPA) was formed during mild oxidation of LDL and was the active compound in mildly oxidized LDL and minimally modified LDL, initiating platelet activation and stimulating endothelial cell stress-fiber and gap formation. Antagonists of the LPA receptor prevented platelet and endothelial cell activation by mildly oxidized LDL. We also found that LPA accumulated in and was the primary platelet-activating lipid of atherosclerotic plaques. Notably, the amount of LPA within the human carotid atherosclerotic lesion was highest in the lipid-rich core, the region most thrombogenic and most prone to rupture. Given the potent biological activity of LPA on platelets and on cells of the vessel wall, our study identifies LPA as an atherothrombogenic molecule and suggests a possible strategy to prevent and treat atherosclerosis and cardiocerebrovascular diseases.

Crystal structure of the Mycobacterium tuberculosis enoyl-ACP reductase, InhA, in complex with NAD+ and a C16 fatty acyl substrate.

Enoyl-ACP reductases participate in fatty acid biosynthesis by utilizing NADH to reduce the trans double bond between positions C2 and C3 of a fatty acyl chain linked to the acyl carrier protein. The enoyl-ACP reductase from Mycobacterium tuberculosis, known as InhA, is a member of an unusual FAS-II system that prefers longer chain fatty acyl substrates for the purpose of synthesizing mycolic acids, a major component of mycobacterial cell walls. The crystal structure of InhA in complex with NAD+ and a C16 fatty acyl substrate, trans-2-hexadecenoyl-(N-acetylcysteamine)-thioester, reveals that the substrate binds in a general "U-shaped" conformation, with the trans double bond positioned directly adjacent to the nicotinamide ring of NAD+. The side chain of Tyr158 directly interacts with the thioester carbonyl oxygen of the C16 fatty acyl substrate and therefore could help stabilize the enolate intermediate, proposed to form during substrate catalysis. Hydrophobic residues, primarily from the substrate binding loop (residues 196-219), engulf the fatty acyl chain portion of the substrate. The substrate binding loop of InhA is longer than that of other enoyl-ACP reductases and creates a deeper substrate binding crevice, consistent with the ability of InhA to recognize longer chain fatty acyl substrates.

Convergent C-glycolipid synthesis via the Ramberg-Bäcklund reaction: active antiproliferative glycolipids.

[formula: see text] A novel methodology has been developed, employing the Ramberg-Bäcklund rearrangement and ionic hydrogenation to synthesize C-glycosides with high stereoselectivity at the anomeric center. The C-glycolipid 14b exhibits antiproliferative properties similar to those of O-glycoside analogue 14a.

Effects of a water-soluble antitumor ether phosphonoinositide, D-myo-inositol 4-(hexadecyloxy)-3(S)-methoxybutanephosphonate (C4-PI), on inositol lipid metabolism in breast epithelial cancer cell lines.

We have demonstrated previously that D-myo-inositol 4-(hexadecyloxy)-3(S)-methoxybutanephosphonate (C4-PI), an isosteric phosphonate analog of phosphatidylinositol developed to inhibit inositol lipid metabolism, was unable to inhibit phosphatidylinositol (PI) 3-kinase activity. We now report the effects of the compound on other aspects of inositol metabolism. We demonstrated that C4-PI inhibits the activity of purified recombinant PI-phospholipase C-beta (PLC-beta) at all concentrations tested; it enhanced the activity of PI-PLC-gamma and PI-PLC-delta at low concentrations (10 microM), while severely inhibiting their activities at higher concentrations. In the breast cancer cell lines MCF-7 (estrogen receptor positive) and MDA-MB-468 (estrogen receptor negative), C4-PI had no effect on the uptake of D-myo-inositol but severely inhibited its incorporation into PI. In spite of the drastic decrease in PI synthesis, C4-PI did not affect the levels of inositol incorporated into phosphatidylinositol 4,5-bisphosphate (PIP2) in the cells. In vitro assays showed that C4-PI inhibited PI synthase activity (inhibition of 35% at 50 microM) but had little effect on PI 4-kinase activity (inhibition of 13% at 150 microM). C4-PI inhibited the proliferation of MCF-7 and MDA-MB-468 cell lines with IC(50) values of 12 and 18 microM. Taken together, the results suggest that the accumulation of [3H]inositol in PIP2 in cells incubated with C4-PI may be due to the inhibition of PIP2 hydrolysis in the cells with no effect on its synthesis. The role of these C4-PI-induced effects in the mechanism of growth inhibition by C4-PI remains to be established.

Evidence for involvement of mitogen-activated protein kinase, rather than stress-activated protein kinase, in potentiation of 1-beta-D-arabinofuranosylcytosine-induced apoptosis by interruption of protein kinase C signaling.

The stress-activated protein kinase (SAPK) and mitogen-activated protein kinase (MAPK) cascades mediate cytotoxic and cytoprotective functions, respectively, in the regulation of leukemic cell survival. Involvement of these signaling systems in the cytotoxicity of 1-beta-D-arabinofuranosylcytosine (ara-C) and modulation of ara-C lethality by protein kinase C PKC inhibition/down-regulation was examined in HL-60 promyelocytic leukemia cells. Exposure to ara-C (10 microM) for 6 hr promoted extensive apoptotic DNA damage and cell death, as well as activation of PKC. This response was accompanied by downstream activation of the SAPK and MAPK cascades. PKC-dependent MAPK activity seemed to limit ara-C action in that the toxicity of ara-C was enhanced by pharmacological reductions of PKC, MAPK, or both. Thus, ara-C action was (1) partially attenuated by diradylglycerols, which stimulated PKC and MAPK, but (2) dramatically amplified by sphingoid bases, which inhibited PKC and MAPK. The cytotoxicity of ara-C also was substantially increased by pharmacological reductions of PKC, including down-regulation of PKC by chronic preexposure to the macrocyclic lactone bryostatin 1 or inhibition of PKC by acute coexposure to the dihydrosphingosine analog safingol. Significantly, both of these manipulations prevented activation of MAPK by ara-C. Moreover, acute disruption of the MAPK module by AMF, a selective inhibitor of MEK1, suppressed both basal and drug-stimulated MAPK activity and sharply increased the cytotoxicity of ara-C, suggesting the direct involvement of MAPK as a downstream antiapoptotic effector for PKC. None of these chemopotentiating agents enhanced ara-CTP formation. Ceramide-driven SAPK activity did not seem to mediate drug-induced apoptosis, given that (1) neutralization of endogenous tumor necrosis factor-alpha with monoclonal antibodies or soluble tumor necrosis factor receptor substantially reduced ceramide generation and SAPK activation by ara-C, whereas the induction of apoptosis was unaffected; (2) pharmacological inhibition of sphingomyelinase by 3-O-methoxysphingomyelin reduced ceramide generation and SAPK activation without limiting the drug's cytotoxicity; and (3) potentiation of ara-C action by bryostatin 1 or safingol was not associated with further stimulation of SAPK. These observations collectively suggest a primary role for decreased MAPK, rather than increased SAPK, in the potentiation of ara-C cytotoxicity by interference with PKC-dependent signaling.