Developmental Regulation by Cytokines of Bone Marrow-Derived Dendritic Cells and Epidermal Langerhans Cells

Dendritic cells (DC) are specialized antigen-presenting cells involved in T cell-mediated immune responses. Differentiation and functional maturation of the DC are now known to be regulated by various cytokines, including TGF-β1. The experiments of this study examined the effect of other cytokines, such as IL-4, IL-10 and IL-6, on the differentiation and maturation of bone marrow (BM)-derived DC (BM-DC) and epidermal Langerhans cells (LC). When IL-6 or IL-10 was added to cultures of BM cells in the presence of GM-CSF, both cytokines, as in the case of TGF-β1, suppressed the maturation of DC in terms of the expression of adhesion and costimulatory molecules and T cell-stimulating activity. In contrast, IL-4 was not suppressive but rather supportive for the differentiation of DC. However, these suppressive cytokines hardly counteracted the maturation-inducing activity of TNF-α when added to cultures of immature DC. In addition, they appeared to block the overmaturation of DC, which is characterized by a loss of MHC class II molecules. Regarding LC maturation in epidermal cell cultures, IL-6 and IL-10 were inhibitory for the expression of CD86 and CD80 in a dose-dependent fashion. Unlike BM-DC, LC maturation was slightly enhanced by TGF-β1. The protein antigen-presentation by LC to Th1 clone was not affected by IL-6, but slightly reduced by IL-10. These results suggest that each cytokine contributes to regulate the differentiation and maturation of DC at a different developmental stage.     

The Src-like adaptor protein regulates GM-CSFR signaling and monocytic dendritic cell maturation

GM-CSF is an important cytokine involved in myeloid differentiation and inflammatory processes. Signaling through the GM-CSFR also plays a critical role in the generation of monocyte-derived dendritic cells (DC). In this article, we report that the Src-like adaptor protein (SLAP) functions as a negative regulator of the GM-CSFR. In bone marrow-derived DC (BM-DC) lacking SLAP and the closely related SLAP2, downregulation of GM-CSFRβ is impaired, leading to enhanced phosphorylation of Jak2 and prolonged activation of Akt and Erk1/2 in response to GM-CSF stimulation. Compared with wild-type bone marrow, SLAP/SLAP2(-/-) bone marrow gave rise to similar numbers of CD11c(+) and CD11b(+) DC, but SLAP/SLAP2(-/-) BM-DC failed to acquire high levels of MHC class II, CD80, and CD86, indicating an impairment in maturation. Furthermore, MHC class II expression in SLAP/SLAP2(-/-) BM-DC was rescued by decreasing GM-CSF concentration, suggesting that enhanced GM-CSF signaling mediates the block in maturation. In addition, SLAP/SLAP2(-/-) BM-DC produced less IL-12 and TNF-α in response to LPS compared with controls and failed to stimulate T cells in an MLR. Ag-specific T cell activation assays showed that SLAP/SLAP2(-/-) BM-DC were less robust at inducing IFN-γ secretion by DO11.10 T cells. These results indicated that SLAP-mediated GM-CSFR regulation is important for the generation of functionally mature monocytic DC.     

Dendritic cells issued in vitro from bone marrow produce PGE(2) that contributes to the immunomodulation induced by antigen-presenting cells.

Given that preliminary work has indicated that prostaglandins can play a role in modulating dendritic cell (DC) functions, we addressed the prostaglandin E(2) (PGE(2)) biosynthetic capacity of mouse DC produced in vitro from bone marrow cells. We observed production of significant amounts of PGE(2), which was reduced by at least 80% when cells were incubated in the presence of indomethacin, a COX-1 preferential inhibitor. Indeed, when tested by Western blot analysis with specific COX-1 and COX-2 antibodies, only COX-1 expression could be detected in the bone marrow (BM)-DC. For lipopolysaccharide (LPS)-treated BM-DC, inhibition of PGE(2) production by indomethacin or by NS-398 (a COX-2-selective inhibitor) used alone was less potent. After LPS treatment of BM-DC, COX-1 and COX-2 expression was potent, and inhibition of PGE(2) synthesis needed the presence of both indomethacin and NS-398. We also observed that exogenous PGE(2) diminished the expression of MHC class II molecules by BM-DC and that prostaglandin and indomethacin had antagonistic effects on cell proliferation during the mixed lymphocyte reaction using BM-DC as stimulatory cells. This assessment of PGE(2) suggests that endogenous PGE(2) produced by DC might play a role as an immunomodulating factor during the immune response. This hypothesis is sustained by the fact that IL-12 production by BM-DC is modulated by exogenous PGE(2) as well as endogenous prostaglandin, since either the addition of exogenous PGE(2) or the presence of LPS (which increases endogenous PGE(2) synthesis) decreases IL-12 production, while NS-398 (which decreases LPS-induced PGE(2) synthesis) increases IL-12 synthesis.     

Lovastatin inhibits G1/S transition of normal human B-lymphocytes independent of apoptosis.

Lovastatin is a potent inhibitor of protein prenylation, and it has been reported to have pleiotropic cellular effects. In the present study we have elucidated the effects of lovastatin on cell cycle progression and apoptosis of normal human B-lymphocytes. When added to B-lymphocytes stimulated with anti-immunoglobulin (anti-mu) and SAC, lovastatin (20 microM) inhibited the cells in the late G1 phase of the cell cycle. Thus, no early activation parameters such as Ca(2+) flux or MYC induction were affected by lovastatin, whereas progression of cells into the second cell cycle as well as DNA synthesis was markedly reduced. We therefore examined the effects of lovastatin on components of the cell cycle machinery responsible for regulating the G1/S transition. We demonstrated that pRB phosphorylation, cdk2 activity needed for this phosphorylation, and the levels of cyclin A, D, and E were inhibited after 24 h of lovastatin treatment, while the levels of p27(Kip1) were elevated. There was no effect on p21(Cip1), cyclin D2, cdk4, and cdk6. These data are consistent with the cells being inhibited by lovastatin between 24 and 32 h into G1. Lovastatin added to stimulated B-cells in late G1 still inhibited the DNA synthesis by 60%, but at this point only minor effects were noted on the cell cycle machinery. We therefore looked for induced apoptosis as an explanation for reduced S-phase entry of the cells. However, despite the ability to enhance the apoptosis of unstimulated B-cells from 48 to 61% as judged by the TUNEL method, lovastatin only marginally affected apoptosis when administered to stimulated B-cells. Thus, it appears that accelerated apoptosis cannot account for the effect of lovastatin on cell cycle progression.     

Low-density lipoprotein-induced expression of interleukin-6, a marker of human mesangial cell inflammation: effects of oxidation and modulation by lovastatin

Low-density lipoprotein (LDL) may contribute to the pathogenesis of glomerulosclerosis by stimulating a mesangial cell inflammatory response. Interleukin-6 (IL-6) is a marker of active inflammation and ongoing glomerular injury. Therefore, we investigated the effects of native and oxidized LDL on human mesangial cell production of IL-6 and a possible modulation of this inflammatory response by lovastatin, which has been shown to ameliorate experimental glomerulosclerosis. Human mesangial cells were exposed for 6 or 24 h to culture medium containing either native LDL alone or a LDL mixture containing 5 or 20% oxidized LDL. We found that native LDL stimulated 6 h mRNA expression and secretion of IL-6. This effect was further enhanced, in a dose-related manner, when mesangial cells were exposed to increasing concentrations of oxidized LDL. Lovastatin markedly inhibited mesangial cell expression of IL-6 mRNA and reduced IL-6 secretion. The inhibitory effects of lovastatin were overridden at least partially by exogenous mevalonate. We conclude that LDL, and particularly oxidized LDL, might contribute to the pathogenesis of glomerular disease by modulating the inflammatory response of human mesangial cells, as assessed by the stimulation of IL-6 expression. Moreover, this inflammatory response can be prevented by lovastatin, providing a potential direct anti-inflammatory mechanism by which HMG-CoA reductase inhibitors may attenuate lipid-induced glomerular injury.     

Human dendritic cells discriminate between viable and killed Toxoplasma gondii tachyzoites: dendritic cell activation after infection with viable parasites results in CD28 and CD40 ligand signaling that controls IL-12-dependent and -independent T cell production of IFN-gamma

We studied how the interaction between human dendritic cells (DC) and Toxoplasma gondii influences the generation of cell-mediated immunity against the parasite. We demonstrate that viable, but not killed, tachyzoites of T. gondii altered the phenotype of immature DC. DC infected with viable parasites up-regulated the expression of CD40, CD80, CD86, and HLA-DR and down-regulated expression of CD115. These changes are indicative of DC activation induced by T. gondii. Viable and killed tachyzoites had contrasting effects on cytokine production. DC infected with viable T. gondii rather than DC that phagocytosed killed parasites induced secretion of high amounts of IFN-gamma by T cells from T. gondii-seronegative donors. IFN-gamma production in response to DC infected with viable parasites required CD28 and CD40 ligand (CD40L) signaling. In addition, this IFN-gamma response was dependent in part on IL-12 secretion. Production of IL-12 p70 occurred after interaction between T cells and DC infected with viable T. gondii, but not after incubation of T cells with DC plus killed tachyzoites. IL-12 synthesis was inhibited by blockade of CD40L signaling. IL-12-independent IFN-gamma production required CD80/CD86-CD28 interaction and, to a lesser extent, CD40-CD40L signaling. Taken together, T. gondii-induced activation of human DC is associated with T cell production of IFN-gamma through CD40-CD40L-dependent release of IL-12 and through CD80/CD86-CD28 and CD40-CD40L signaling that mediate IFN-gamma secretion even in the absence of bioactive IL-12.     

Prostaglandin I2 analogs inhibit proinflammatory cytokine production and T cell stimulatory function of dendritic cells

Signaling through the PGI(2) receptor (IP) has been shown to inhibit inflammatory responses in mouse models of respiratory syncytial viral infection and OVA-induced allergic responses. However, little is known about the cell types that mediate the anti-inflammatory function of PGI(2.) In this study, we determined that PGI(2) analogs modulate dendritic cell (DC) cytokine production, maturation, and function. We report that PGI(2) analogs (iloprost, cicaprost, treprostinil) differentially modulate the response of murine bone marrow-derived DC (BMDC) to LPS in an IP-dependent manner. The PGI(2) analogs decreased BMDC production of proinflammatory cytokines (IL-12, TNF-alpha, IL-1alpha, IL-6) and chemokines (MIP-1alpha, MCP-1) and increased the production of the anti-inflammatory cytokine IL-10 by BMDCs. The modulatory effect was associated with IP-dependent up-regulation of intracellular cAMP and down-regulation of NF-kappaB activity. Iloprost and cicaprost also suppressed LPS-induced expression of CD86, CD40, and MHC class II molecules by BMDCs and inhibited the ability of BMDCs to stimulate Ag-specific CD4 T cell proliferation and production of IL-5 and IL-13. These findings suggest that PGI(2) signaling through the IP may exert anti-inflammatory effects by acting on DC.     

Lovastatin is a potent inhibitor of cholecystokinin secretion in endocrine tumor cells in culture

Lovastatin prevents isoprene synthesis thereby affecting the structural organization of proteins involved in protein transport and secretion. Lovastatin at 1 microM decreases CCK 8 secretion by over 50% in WE cells and in CCK 8 expressing AtT20 cells. At 10 microM CCK 8 secretion was inhibited by two thirds and at 100 microM, cytotoxic effects were observed in both cell types. Addition of mevalonate does not restore CCK secretion and stimulation of secretion by forskolin is also partially inhibited. Cellular content of CCK 8 and pro-CCK were not altered in either of these cell lines except at 100 microM lovastatin. Our results clearly demonstrate that lovastatin at 1 microM strongly inhibits CCK 8 secretion at multiple levels while having little or no effect on its synthesis. This effect on secretion may be partly responsible for the adverse gastrointestinal side effects of lovastatin in patients.     

Cooperative induction of a tolerogenic dendritic cell phenotype by cytokines secreted by pancreatic carcinoma cells

Ag presentation by dendritic cells (DC) is essential to effective antitumor T cell responses in cancer patients. Depending on their origin, maturation state, and the ambient cytokine milieu, DC can differentiate into distinct subpopulations, which preferentially either induce Th1 cell activation (CD11c+,CD123- myeloid DC (MDC)) or immunosuppressive T cell development (CD11c-,CD123+ plasmacytoid DC (PDC)). The present study was undertaken to characterize the effects of pancreatic carcinoma cell-derived cytokines on immature monocyte-derived DC (iMo-DC) in vitro and in vivo. Medium conditioned by human pancreatic carcinoma cells inhibited iMo-DC proliferation, expression of costimulatory molecules (CD80 and CD40) and of HLA-DR, and functional activity as assessed by MLR and IL-12p70 production. iMo-DC generated from pancreatic carcinoma patients in advanced stages of the disease similarly showed decreased levels of HLA-DR expression and reduced ability to stimulate MLR in response to CD40L and IFN-gamma. Moreover, in tumor-patient peripheral blood, the ratio of MDC to PDC cells was lower than in healthy controls due to reduced numbers of MDC CD11c+ cells. Importantly, rather than a single cytokine, a combination of tumor-derived cytokines was responsible for these effects; these were primarily TGF-beta, IL-10, and IL-6, but not vascular endothelial growth factor. In summary, we have identified an array of pancreatic carcinoma-derived cytokines that cooperatively affect iMo-DC activation in a manner consistent with ineffective antitumor immune responses.     

Lovastatin inhibits the growth and survival pathway of phosphoinositide 3-kinase/protein kinase B in immortalized rat brain neuroblasts

We previously showed that lovastatin, an HMG-CoA reductase inhibitor, suppresses cell growth by inducing apoptosis in rat brain neuroblasts. Our aim was to study intracellular signalling induced by lovastatin in neuroblasts. Lovastatin significantly decreases the phosphoinositide 3-kinase (PI3-K) activity in a concentration-dependent manner. Expression of p85 subunit and its association with phosphotyrosine-containing proteins are unaffected by lovastatin. Lovastatin decreases protein kinase B (PKB)/Akt phosphorylation, and its downstream effectors, p70^S6K and the eukaryotic initiation factor 4E (eIF4E) regulatory protein 1, 4E-BP1, in a concentration-dependent manner, and reduces p70^S6K expression. Lovastatin effects are fully prevented with mevalonate. Only the highest dose of PI3-K inhibitors that significantly reduce PI3-K kinase activity induces apoptosis in neuroblasts but to a lower degree than lovastatin. In summary, this work shows that treatment of brain neuroblasts with lovastatin leads to an inhibition of the main pathway that controls cell growth and survival, PI3-K/PKB and the subsequent blockade of downstream proteins implicated in the regulation of protein synthesis. This work suggests that inactivation of the antiapoptotic PI3-K appears insufficient to induce the degree of neuroblasts apoptosis provoked by lovastatin, which must necessarily involve other intracellular pathways. These findings might contribute to elucidate the molecular mechanisms of some statins effects in the central nervous system.     

Effect of lovastatin on acyl-CoA: cholesterol O-acyltransferase (ACAT) activity and the basolateral-membrane secretion of newly synthesized lipids by CaCo-2 cells.

Lovastatin, a potent competitive inhibitor of 3-hydroxy-3-methylglutaryl-CoA (HMG-CoA) reductase activity, was used to study the regulation of cholesterol metabolism and the basolateral-membrane secretion of triacylglycerol and cholesterol in the human intestinal cell line CaCo-2. At 0.1 microgram/ml, lovastatin decreased 3H2O incorporation into cholesterol by 71%. In membranes prepared from cells incubated with lovastatin for 18 h, HMG-CoA reductase activity was induced 4-8-fold. Mevalonolactone prevented this induction. In intact cells, lovastatin (10 micrograms/ml) decreased cholesterol esterification by 50%. The reductase inhibitor decreased membrane acyl-CoA:cholesterol O-acyltransferase (ACAT) activity by 50% at 5 micrograms/ml. ACAT inhibition by lavastatin was not reversed by adding excess of cholesterol or fatty acyl-CoA to the assay. Lovastatin, in the presence or absence of mevalonolactone, decreased the basolateral secretion of newly synthesized cholesteryl esters and triacylglycerols. Lovastatin also inhibited the esterification of absorbed cholesterol and the secretion of this newly synthesized cholesteryl ester. Lovastatin is a potent inhibitor of cholesterol synthesis in CaCo-2 cells. Moreover, it is a direct inhibitor of ACAT activity, independently of its effect on HMG-CoA reductase and cholesterol synthesis.     

Effects of 5-azacytidine and trichostatin A on dendritic cell maturation

Maturation of dendritic cells (DC) towards functional antigen-presenting cells is a complex process, the regulation of which may also involve epigenetic mechanisms. Thus, it is of interest to investigate how gene expression changes during DC maturation can be influenced with epigenetic agents, such as DNA methyltransferase or histone deacetylase inhibitors. Here, we document the effects of DNA methyltransferase inhibitor 5-azacytidine (5AC) and histone deacetylase inhibitor trichostatin A (TSA) on the murine bone marrow-derived, as well as on the human monocyte-derived DC maturation. The major impact of 5AC and TSA on the DC maturation process consisted in the inhibition of unmethylated CpG oligodeoxynucleotide (CpG ODN) 1826 or LPS-induced activation of pro- and anti-inflammatory cytokine gene expression activation. In the in vitro studies, TSA but not 5AC significantly reduced the capacity of the peptide-pulsed DC to induce total spleen as well as CD8(+) or CD4(+) cell proliferation. IFNγ production by the specific CD4(+) spleen cells co-cultured with TSA- but not with 5AC-treated DC was lower, as compared to the cytokine production after co-cultivation with untreated mature DC. Collectively, these results demonstrate the potential of epigenetic agents, which are under intensive investigation as promising anti-tumour agents, to hamper the immune response induction through their inhibitory effects on DC.     

Deconstructing tick saliva: non-protein molecules with potent immunomodulatory properties

Dendritic cells (DCs) are powerful initiators of innate and adaptive immune responses. Ticks are blood-sucking ectoparasite arthropods that suppress host immunity by secreting immunomodulatory molecules in their saliva. Here, compounds present in Rhipicephalus sanguineus tick saliva with immunomodulatory effects on DC differentiation, cytokine production, and costimulatory molecule expression were identified. R. sanguineus tick saliva inhibited IL-12p40 and TNF-α while potentiating IL-10 cytokine production by bone marrow-derived DCs stimulated by Toll-like receptor-2, -4, and -9 agonists. To identify the molecules responsible for these effects, we fractionated the saliva through microcon filtration and reversed-phase HPLC and tested each fraction for DC maturation. Fractions with proven effects were analyzed by micro-HPLC tandem mass spectrometry or competition ELISA. Thus, we identified for the first time in tick saliva the purine nucleoside adenosine (concentration of ～110 pmol/μl) as a potent anti-inflammatory salivary inhibitor of DC cytokine production. We also found prostaglandin E(2) (PGE(2) ～100 nM) with comparable effects in modulating cytokine production by DCs. Both Ado and PGE(2) inhibited cytokine production by inducing cAMP-PKA signaling in DCs. Additionally, both Ado and PGE(2) were able to inhibit expression of CD40 in mature DCs. Finally, flow cytometry analysis revealed that PGE(2), but not Ado, is the differentiation inhibitor of bone marrow-derived DCs. The presence of non-protein molecules adenosine and PGE(2) in tick saliva indicates an important evolutionary mechanism used by ticks to subvert host immune cells and allow them to successfully complete their blood meal and life cycle.     

Exogenous dendritic cell homing to draining lymph nodes can be boosted by mast cell degranulation

Dendritic cells (DCs), as potent antigen presenting cells, are increasingly used for immunotherapeutic approaches, predominantly in oncology. Low efficiency of injected Ag-pulsed DC homing to draining lymph nodes (DLNs) is one of the factors that affect the efficacy of therapy. As Langerhans cell emigration was enhanced after skin mast cell degranulation, we investigated the effect of local mast cell activation on exogenous bone marrow-derived DCs (BM-DCs) homing to DLNs. Product of activated MC/9 mast cells enhanced chemotaxis of BM-DCs to CCL21 in vitro. Intradermal injection of compound 48/80 (c48/80) induced local skin mast cell obvious degranulation and boosted exogenous BM-DC homing to DLNs. Both Ag-specific lymphocyte proliferation and TH1/TH2 cytokine production increased after HBsAg-pulsed BM-DC was injected into c48/80 pretreated mice. These results suggest that transferred DC homing to DLNs promoted by local mast cell degranulation may have potential application to improve DC-based immunotherapy.     

Lovastatin inhibits human B lymphoma cell proliferation by reducing intracellular ROS and TRPC6 expression

Clinical evidence suggests that statins reduce cancer incidence and mortality. However, there is lack of in vitro data to show the mechanism by which statins can reduce the malignancies of cancer cells. We used a human B lymphoma Daudi cells as a model and found that lovastatin inhibited, whereas exogenous cholesterol (Cho) stimulated, proliferation cell cycle progression in control Daudi cells, but not in the cells when transient receptor potential canonical 6 (TRPC6) channel was knocked down. Lovastatin decreased, whereas Cho increased, the levels of intracellular reactive oxygen species (ROS) respectively by decreasing or increasing the expression of p47-phox and gp91-phox (NOX2). Reducing intracellular ROS with either a mimetic superoxide dismutase (TEMPOL) or an NADPH oxidase inhibitor (apocynin) inhibited cell proliferation, particularly in Cho-treated cells. The effects of TEMPOL or apocynin were mimicked by inhibition of TRPC6 with SKF-96365. Lovastatin decreased TRPC6 expression and activity via a Cho-dependent mechanism, whereas Cho increased TRPC6 expression and activity via an ROS-dependent mechanism. Consistent with the fact that TRPC6 is a Ca^2 +-permeable channel, lovastatin decreased, but Cho increased, intracellular Ca^2 + also via ROS. These data suggest that lovastatin inhibits malignant B cell proliferation by reducing membrane Cho, intracellular ROS, TRPC6 expression and activity, and intracellular Ca^2 +.     

Altered dendritic cells (DC) might be responsible for regulatory T cell imbalance and autoimmunity in nonobese diabetic (NOD) mice

Nonobese diabetic (NOD) mice spontaneously develop diabetes, an auto-immune disease characterized by the destruction of insulin-secreting beta-cells by autoreactive T cells. Defects in development and/or functions of dendritic cells (DC) might be critical in eliciting the auto-immune reaction to beta cells in this model. In this paper, DC differentiation in NOD mice was investigated in vitro using bone marrow-derived progenitors (BM-DC) in the presence of GM-CSF and IL-4 or spleen-derived progenitors in the presence of GM-CSF and early acting cytokines such as Flt-3L and IL-6 (SPL-DC). In both culture systems, the absolute number of NOD DC generated was strongly reduced as compared to control strains. In addition, both BM-DC and SPL-DC from NOD mice show defective differentiation into mature DC in conventional culture conditions as indicated by low expression of MHC class II and CD80 molecules among CD11c positive cells and low capacity to stimulate allogeneic T cells. However, DC achieved full maturation when exposed to LPS, except for MHC class II expression that remained decreased. Ex vivo analysis confirmed an unusual phenotype of NOD DC. Both sets of results are thus consistent with a specific defect of DC maturation in these mice.     

Normal immune function of monocyte-derived dendritic cells from HIV-infected individuals: implications for immunotherapy.

Dendritic cells (DC) are the most potent cells involved in the generation of primary and secondary immune responses. To assess the feasibility of using autologous DC as immunotherapy for HIV disease, we analyzed a variety of immune parameters using DC isolated from HIV-infected (HIV+) individuals, as well as DC obtained from HIV-uninfected (HIV-) individuals infected in vitro with HIV. After stimulation with recombinant CD40 ligand (CD40LT), cytokine and beta-chemokine production were similar by DC from HIV- donors infected in vitro with the CCR5-using HIV Ba-L strain (n = 8) compared with uninfected DC from the same donors. Production of beta-chemokines, but not of cytokines, was increased by a CXCR4-using IIIB strain-infected DC (n = 7). Stimulation of HIV-infected DC with CD40LT decreased infection in Ba-L-infected DC, but had no effect on IIIB-infected DC. Consistent with this finding, CD40LT down-regulated CCR5 and up-regulated CXCR4 expression on DC. Monocyte-derived DC were also propagated from 15 HIV+ and 13 HIV- donors. They exhibited similar expression of costimulatory molecules and produced similar amounts of IL-12, IL-10, and beta-chemokines, following stimulation. By contrast, stimulated PBMC from HIV+ patients exhibited decreased IL-12 and increased IL-10 production. In summary, phenotype, cytokine secretion, and beta-chemokine production by DC from HIV+ individuals were normal. These cells may prove useful in boosting cellular immune responses in HIV+ individuals.     

Lovastatin enhances phenylbutyrate-induced MR-visible glycerophosphocholine but not apoptosis in DU145 prostate cells

In this study the effects of lovastatin on DU145 prostate cancer cells treated with phenylbutyrate (PB) was investigated in order to determine the NMR-detectable metabolic changes resulting from the cooperative activity of these two agents. DU145 cells were perfused with PB in the presence or absence of 10 microM of the HMG-CoA reductase inhibitor lovastatin, and the results monitored by 31P and diffusion-weighted 1H NMR spectroscopy. Lovastatin had additive effects on the PB-induced NMR-visible total choline in 1H spectra, and glycerophosphocholine in 31P spectra but no significant effect on NMR-visible lipid. Moreover, lovastatin had no effect on the ability of PB to either promote the formation of oil red O-detectable lipid droplets or arrest the cell cycle. The most remarkable observations from these studies were that lovastatin enhanced the increase in glycerophosphocholine while reversing late markers of apoptosis and the loss of NTP caused by PB. These results identify a branch point separating the neutral lipid production and the apoptotic cell death caused by the actions of differentiating agents.