Suppression of ceramide-mediated apoptosis by HSP70.

Ceramide has been known as an important second messenger in programmed cell death (apoptosis) which is induced by various stimuli such as the tumor necrosis factor-alpha (TNF-alpha), Fas ligand, and environmental stresses such as UV-irradiation and heat shock. Although the precise molecular mechanism of apoptosis is not fully understood, ceramide generated by sphingomyelinase (SMase) mediates the activation of several downstream molecules that are implicated in the regulation of apoptosis. Here, we show that stress-inducible heat shock protein 70 (Hsp70) prevents apoptosis induced by increased level of intracellular ceramide. In T-cell hybridoma DO11.10, we examined the effect of Hsp70 on apoptosis mediated by TNF-alpha, Fas ligation, SMase, and C2-ceramide, all of which elevate intracellular ceramide levels. Hsp70 not only markedly reduced internucleosomal DNA fragmentation, but also enhanced cell viability measured by the Trypan blue dye exclusion test. Similarly, the ceramide-induced c-jun amino-terminal kinase (JNK/SAPK) activation is impaired in cells overexpressing Hsp70. These data strongly suggest that hsp70 functions as a regulator of apoptosis downstream of ceramide.     

Docosahexaenoic acid down-regulates phenobarbital-induced cytochrome P450 2B1 gene expression in rat primary hepatocytes via the sphingomyelinase/ceramide pathway

Docosahexaenoic acid (DHA) regulates the expression of cytochrome P450 2B1 (CYP 2B1) in rat primary hepatocytes in response to xenobiotics. Ceramide, a lipid signaling molecule, is involved in various physiological processes and can be generated by the hydrolysis of sphingomyelin via sphingomyelinase (SMase). DHA activates SMase and increases ceramide formation in vitro. Ceramides differentially enhance adenylyl cyclase activity in vitro depending on the chain length of their fatty acids. In addition, the cAMP-dependent PKA pathway down-regulates CYP 2B1 expression induced by phenobarbital (PB). In the present study, we determined the effect of DHA on SMase transactivation and the downstream pathway in CYP 2B1 expression induced by PB. SMase was activated by DHA 2 h after treatment, and D609 (an SMase inhibitor) attenuated the inhibition of PB-induced CYP 2B1 expression by DHA. Ceramide formation reached a maximum 3 h after DHA administration. C2-ceramide dose-dependently inhibited PB-induced CYP 2B1 expression and increased intracellular cAMP concentrations. SQ22536 (an adenylyl cyclase inhibitor) and H89 (a PKA-specific inhibitor) partially reversed the inhibition of PB-induced CYP 2B1 expression by C2-ceramide. These results suggest that stimulation of SMase, generation of ceramide and activation of the cAMP-dependent PKA pathway are involved in the inhibition exerted by DHA.     

Neutral sphingomyelinase 2 induces dopamine uptake through regulation of intracellular calcium

Ceramide serves as a second messenger produced from sphingomyelin by the activation of sphingomyelinase (SMase). Here, we suggest that neutral SMase 2 (nSMase2) may regulate dopamine (DA) uptake. nSMase2 siRNA-transfected PC12 cells showed lower levels of nSMase activity and ceramide than scramble siRNA-transfected and control cells. Interestingly, transfection of nSMase2 siRNA or pretreatment with the nSMase2-specific inhibitor GW4869 resulted in decreased DA uptake. Reciprocally, exposure of PC12 cells to cell-permeable C₆-ceramide induced a concentration-dependent increase in DA uptake. Removal of extracellular calcium by EGTA increased DA uptake in scramble-transfected and control cells, but not in nSMase2 siRNA-transfected or GW4869-pretreated cells. Moreover, siRNA-transfected cells showed higher levels of intracellular calcium than scramble cells, while C₆-ceramide treatment resulted in decreased intracellular calcium compared to vehicle treatment alone. Taken together, these data suggest that nSMase2 may increase DA uptake through inducing ceramide production and thereby decreasing intracellular calcium levels.     

Ceramide Induces Non-Apoptotic Cell Death in Human Glioma Cells

Ceramide causes either apoptosis or non-apoptotic cell death depending on model system and experimental conditions. The present study was undertaken to examine the effect of ceramide on cell viability and its molecular events leading to cell death in A172 human glioma cells. Ceramide induced cell death in a dose-dependent manner and the cell death was dependent on generation of reactive oxygen species and lipid peroxidation. TUNEL assay, Hoechst 33258 staining, and flow cytometric analysis did not show typical apoptotic morphological features. Ceramide caused phosphorylation of extracellular signal-regulated kinase (ERK) and p38, but the cell death was not affected by inhibitors of MAPK subfamilies. Ceramide caused ATP depletion without loss of mitochondrial membrane potential. Ceramide did not induce caspase activation and ceramide-induced cell death was also not altered by inhibitors of caspase activation. Transfection of dominant inhibitory mutant of IκBα (S32A/36A) and pretreatment of pyrrolidinedithiocarbamate, an inhibitor of NF-κB, enhanced ceramide-induced cell death. These results indicate that ceramide causes non-apoptotic, caspase-independent cell death by inducing reactive oxygen species generation in A172 human glioma cells. NF-κB is involved in the regulation of ceramide-induced cell death in human glioma cells.     

Ceramide enables fas to cap and kill

Recent studies suggest that trimerization of Fas is insufficient for apoptosis induction and indicate that super-aggregation of trimerized Fas might be prerequisite. For many cell surface receptors, cross-linking by multivalent ligands or antibodies induces their lateral segregation within the plasma membrane and co-localization into "caps" on one pole of the cell. In this study, we show that capping of Fas is essential for optimal function and that capping is ceramide-dependent. In Jurkat T lymphocytes and in primary cultures of hepatocytes, ceramide elevation was detected as early as 15-30 s and peaked at 1 min after CH-11 and Jo2 anti-Fas antibody treatment, respectively. Capping was detected 30 s after Fas ligation, peaked at 2 min, and was maintained at a lower level for as long as 30 min in both cell types. Ceramide generation appeared essential for capping. Acid sphingomyelinase -/- hepatocytes were defective in Jo2-induced ceramide generation, capping, and apoptosis, and nanomolar concentrations of C(16)-ceramide restored these events. To further explore the role of ceramide in capping of Fas, we employed FLAG-tagged soluble Fas ligand (sFasL), which binds trimerized Fas but is unable to induce capping or apoptosis in Jurkat cells. Cross-linking of sFasL with M2 anti-FLAG antibody induced both events. Pretreatment of cells with natural C(16)-ceramide bypassed the necessity for forced antibody cross-linking and enabled sFasL to cap and kill. The presence of intact sphingolipid-enriched membrane domains may be essential for Fas capping since their disruption with cholesterol-depleting agents abrogated capping and prevented apoptosis. These data suggest that capping is a ceramide-dependent event required for optimal Fas signaling in some cells.     

Regulation of phospholipase D activity and ceramide production in daunorubicin-induced apoptosis in A-431 cells

We demonstrated here that daunorubicin induced apoptosis in A-431 cells, a human epidermoid carcinoma cell line. Treatment of cells with daunorubicin induced chromatin condensation, nuclear fragmentation, internucleosomal DNA degradation, and the proteolytic cleavage of PKC-delta and poly(ADP-ribose) polymerase in A-431 cells. Daunorubicin, as well as sphingomyelinase (SMase) and the exogenous cell-permeable ceramide analogue C(2)-ceramide, inhibited phospholipase D activity stimulated by phorbol 12-myristate 13-acetate or epidermal growth factor (EGF). Like ceramide, daunorubicin also decreased EGF-induced diacylglycerol generation. However, no increase in ceramide level was observed in daunorubicin-induced apoptosis in A-431 cells. Moreover, treatment of A-431 cells with exogenous cell-permeable C(2)-ceramide or SMase did not induce apoptosis. These results indicate that daunorubicin induces apoptosis in A-431 cells via a mechanism that does not involve increased ceramide formation.     

Reduction of airway anion secretion via CFTR in sphingomyelin pathway

The present study concerns the involvement of the ceramide produced through sphingomyelinase (SMase)-mediated catalysis in airway anion secretion of Calu-3 cells. Short-circuit current (Isc) measurement revealed that isoproterenol (ISO, 0.1 microM)-induced anion secretion was prevented by pretreatment with SMase (0.3 U/ml, for 30 min) from the basolateral but not the apical side, although basal and 1-ethyl-2-benzimidazolinone (1-EBIO, a Ca2+-activated K+ channel opener)-induced Isc were unaffected. The effects of SMase were reproduced in responses to forskolin (20 microM) or 8-bromo-cAMP (2 mM). C2-ceramide, a cell-permeable analog, also repressed the 8-bromo-cAMP-induced responses. Nystatin permeabilization studies confirmed that the SMase- and C2-ceramide-induced repressions were due to hindrance of augmentation of cystic fibrosis transmembrane conductance regulator (CFTR)-mediated conductance across the apical membrane. Further, SMase failed to influence K+ conductance across the basolateral membrane. These results suggest that the ceramide originating from basolateral sphingomyelin acts on activated CFTR from the cytosolic side, hindering anion secretion.     

Ceramide channels increase the permeability of the mitochondrial outer membrane to small proteins

Ceramides are known to play a major regulatory role in apoptosis by inducing cytochrome c release from mitochondria. We have previously reported that C(2)- and C(16)-ceramide, but not dihydroceramide, form large channels in planar membranes (Siskind, L. J., and Colombini, M. (2001) J. Biol. Chem. 275, 38640-38644). Here we show that ceramides do not trigger a cytochrome c secretion or release mechanism, but simply raise the permeability of the mitochondrial outer membrane, via ceramide channel formation, to include small proteins. Exogenously added reduced cytochrome c was able to freely permeate the mitochondrial outer membrane with entry to and exit from the intermembrane space facilitated by ceramides in a dose- and time-dependent manner. The permeability pathways were eliminated upon removal of C(2)-ceramide by bovine serum albumin, thus ruling out a detergent-like effect of C(2)-ceramide on membranes. Ceramide channels were not specific to cytochrome c, as ceramides induced release of adenylate kinase, but not fumerase from isolated mitochondria, showing some specificity of these channels for the outer mitochondrial membrane. SDS-PAGE results show that ceramides allow release of intermembrane space proteins with a molecular weight cut-off of about 60,000. These results indicate that the ceramide-induced membrane permeability increases in isolated mitochondria are via ceramide channel formation and not a release mechanism, as the channels that allow cytochrome c to freely permeate are reversible, and are not specific to cytochrome c.     

Inhibition of Neutral Sphingomyelinase Activation and Ceramide Formation by Glutathione in Hypoxic PC12 Cell Death

Reduced glutathione (GSH) and N-acetylcysteine (NAC), but not other antioxidative or reducing agents, were found to inhibit cell death, both apoptosis and necrosis, induced by hypoxia in naive and nerve growth factor-differentiated PC12 cells. The level of intracellular total GSH decreased time-dependently during hypoxia, but exogenously added GSH prevented such a decrease in GSH. Pretreatment of cells with exogenous GSH or NAC resulted in inhibition of both neutral sphingomyelinase (SMase) activation and ceramide formation during hypoxia. In the in vitro assay system, neutral SMase activity was inhibited dose-dependently by GSH and NAC. Activation of caspase-3 induced by hypoxia was also inhibited by either GSH or NAC. NAC but not GSH inhibited caspase-3 activation induced by C2-ceramide. These results suggest that GSH protects cells from hypoxic injury by direct inhibition of neutral SMase activity and ceramide formation, resulting in inhibition of caspase-3 activation, and that NAC exerts an additional inhibitory effect(s) downstream of ceramide.     

Ceramide-induced formation of ROS and ATP depletion trigger necrosis in lymphoid cells

In lymphocytes, Fas activation leads to both apoptosis and necrosis, whereby the latter form of cell death is linked to delayed production of endogenous ceramide and is mimicked by exogenous administration of long- and short-chain ceramides. Here molecular events associated with noncanonical necrotic cell death downstream of ceramide were investigated in A20 B lymphoma and Jurkat T cells. Cell-permeable, C6-ceramide (C6), but not dihydro-C6-ceramide (DH-C6), induced necrosis in a time- and dose-dependent fashion. Rapid formation of reactive oxygen species (ROS) within 30 min of C6 addition detected by a dihydrorhodamine fluorescence assay, as well as by electron spin resonance, was accompanied by loss of mitochondrial membrane potential. The presence of N-acetylcysteine or ROS scavengers like Tiron, but not Trolox, attenuated ceramide-induced necrosis. Alternatively, adenovirus-mediated expression of catalase in A20 cells also attenuated cell necrosis but not apoptosis. Necrotic cell death observed following C6 exposure was associated with a pronounced decrease in ATP levels and Tiron significantly delayed ATP depletion in both A20 and Jurkat cells. Thus, apoptotic and necrotic death induced by ceramide in lymphocytes occurs via distinct mechanisms. Furthermore, ceramide-induced necrotic cell death is linked here to loss of mitochondrial membrane potential, production of ROS, and intracellular ATP depletion.     

Cell cycle arrest of hematopoietic cell lines after treatment with ceramide is commonly associated with retinoblastoma activation

BACKGROUND: Leukaemia cells differ from their normal counterparts in that their ability to properly regulate survival, proliferation, differentiation, and apoptosis is aberrant. Understanding the molecular mechanisms controlling cell proliferation and developing therapeutic strategies to correct nonfunctional regulatory mechanisms are emerging areas of medical research. Ceramide, a metabolite of membrane sphingomyelin hydrolysis, has recently emerged as a key regulator of cellular proliferation, differentiation, and apoptosis in leukaemia cells. METHODS: Leukaemia cell lines were treated with a biologically active analogue of ceramide, C(2)-ceramide. Cell cycle status was assessed flow cytometrically using propidium iodide. Induction of apoptosis was confirmed by annexin V staining of externalised phosphatidylserine and retinoblastoma activation was determined by Western blotting. RESULTS: C(2)-ceramide induced activation of retinoblastoma tumour suppressor protein, G(0)/G(1) cell cycle arrest, or apoptosis in leukaemia cell lines. In addition, these effects differed depending upon cell type, thus confirming the pleiotropic nature of the ceramide signalling pathway. Most cells studied responded to exogenous C(2)-ceramide by entering growth arrest, evidently resulting from activation of retinoblastoma protein, and by displaying some degree of apoptosis. CONCLUSIONS: Taken together, these findings suggest that signalling via ceramide has novel therapeutic applications for treatment of leukaemia.     

Involvement of the acid sphingomyelinase pathway in uva-induced apoptosis

The sphingomyelin-ceramide pathway is an evolutionarily conserved ubiquitous signal transduction system that regulates many cell functions including apoptosis. Sphingomyelin (SM) is hydrolyzed to ceramide by different sphingomyelinases. Ceramide serves as a second messenger in mediating cellular effects of cytokines and stress. In this study, we find that acid sphingomyelinase (SMase) activity was induced by UVA in normal JY lymphoblasts but was not detectable in MS1418 lymphoblasts from Niemann-Pick type D patients who have an inherited deficiency of acid SMase. We also provide evidence that UVA can induce apoptosis by activating acid SMase in normal JY cells. In contrast, UVA-induced apoptosis was inhibited in MS1418 cells. Exogenous SMase and its product, ceramide (10-40 micrometer), induced apoptosis in JY and MS1418 cells, but the substrate of SMase, SM (20-80 micrometer), induced apoptosis only in JY cells. These results suggest that UVA-induced apoptosis by SM is dependent on acid SMase activity. We also provide evidence that induction of apoptosis by UVA may occur through activation of JNKs via the acid SMase pathway.     

Ceramide-mediated macroautophagy involves inhibition of protein kinase B and up-regulation of beclin 1

The sphingolipid ceramide is involved in the cellular stress response. Here we demonstrate that ceramide controls macroautophagy, a major lysosomal catabolic pathway. Exogenous C(2)-ceramide stimulates macroautophagy (proteolysis and accumulation of autophagic vacuoles) in the human colon cancer HT-29 cells by increasing the endogenous pool of long chain ceramides as demonstrated by the use of the ceramide synthase inhibitor fumonisin B(1). Ceramide reverted the interleukin 13-dependent inhibition of macroautophagy by interfering with the activation of protein kinase B. In addition, C(2)-ceramide stimulated the expression of the autophagy gene product beclin 1. Ceramide is also the mediator of the tamoxifen-dependent accumulation of autophagic vacuoles in the human breast cancer MCF-7 cells. Monodansylcadaverine staining and electron microscopy showed that this accumulation was abrogated by myriocin, an inhibitor of de novo synthesis ceramide. The tamoxifen-dependent accumulation of vacuoles was mimicked by 1-phenyl-2-decanoylamino-3-morpholino-1-propanol, an inhibitor of glucosylceramide synthase. 1-Phenyl-2-decanoylamino-3-morpholino-1-propanol, tamoxifen, and C(2)-ceramide stimulated the expression of beclin 1, whereas myriocin antagonized the tamoxifen-dependent up-regulation. Tamoxifen and C(2)-ceramide interfere with the activation of protein kinase B, whereas myriocin relieved the inhibitory effect of tamoxifen. In conclusion, the control of macroautophagy by ceramide provides a novel function for this lipid mediator in a cell process with major biological outcomes.     

Tumor necrosis factor-alpha, sphingomyelinase, and ceramide inhibit store-operated calcium entry in thyroid FRTL-5 cells

Tumor necrosis factor alpha (TNF-alpha) is a potent inhibitor of proliferation in several cell types, including thyroid FRTL-5 cells. As intracellular free calcium ([Ca2+]i) is a major signal in activating proliferation, we investigated the effect of TNF-alpha on calcium fluxes in FRTL-5 cells. TNF-alpha per se did not modulate resting [Ca2+]i. However, preincubation (10 min) of the cells with 1-100 ng/ml TNF-alpha decreased the thapsigargin (Tg)-evoked store-operated calcium entry in a concentration-dependent manner. TNF-alpha did not inhibit the mobilization of sequestered calcium. To investigate whether the effect of TNF-alpha on calcium entry was mediated via the sphingomyelinase pathway, the cells were pretreated with sphingomyelinase (SMase) prior to stimulation with Tg. SMase inhibited the Tg-evoked calcium entry in a concentration-dependent manner. Furthermore, an inhibition of calcium entry was obtained after preincubation of the cells with the membrane-permeable C2-ceramide and C6-ceramide analogues. The inactive ceramides dihydro-C2 and dihydro-C6 showed only marginal effects. Neither SMase, C2-ceramide, nor C6-ceramide affected the release of sequestered calcium. C2- and C6-ceramide also decreased the ATP-evoked calcium entry, without affecting the release of sequestered calcium. The effect of TNF-alpha and SMase was inhibited by the kinase inhibitor staurosporin and by the protein kinase C (PKC) inhibitor calphostin C but not by down-regulation of PKC. However, we were unable to measure a significant activation of PKC using TNF-alpha or C6-ceramide. The effect of TNF-alpha was not mediated via activation of either c-Jun N-terminal kinase or p38 kinase. We were unable to detect an increase in the ceramide (or sphingosine) content of the cells after stimulation with TNF-alpha for up to 30 min. Thus, one mechanism of action of TNF-alpha, SMase, and ceramide on thyroid FRTL-5 cells is to inhibit calcium entry.     

Subtype-specific translocation of the delta subtype of protein kinase C and its activation by tyrosine phosphorylation induced by ceramide in HeLa cells

We investigated the functional roles of ceramide, an intracellular lipid mediator, in cell signaling pathways by monitoring the intracellular movement of protein kinase C (PKC) subtypes fused to green fluorescent protein (GFP) in HeLa living cells. C(2)-ceramide but not C(2)-dihydroceramide induced translocation of delta PKC-GFP to the Golgi complex, while alpha PKC- and zeta PKC-GFP did not respond to ceramide. The Golgi-associated delta PKC-GFP induced by ceramide was further translocated to the plasma membrane by phorbol ester treatment. Ceramide itself accumulated to the Golgi complex where delta PKC was translocated by ceramide. Gamma interferon also induced the delta PKC-specific translocation from the cytoplasm to the Golgi complex via the activation of Janus kinase and Mg(2+)-dependent neutral sphingomyelinase. Photobleaching studies showed that ceramide does not evoke tight binding of delta PKC-GFP to the Golgi complex but induces the continuous association and dissociation of delta PKC with the Golgi complex. Ceramide inhibited the kinase activity of delta PKC-GFP in the presence of phosphatidylserine and diolein in vitro, while the kinase activity of delta PKC-GFP immunoprecipitated from ceramide-treated cells was increased. The immunoprecipitated delta PKC-GFP was tyrosine phosphorylated after ceramide treatment. Tyrosine kinase inhibitor abolished the ceramide-induced activation and tyrosine phosphorylation of delta PKC-GFP. These results suggested that gamma interferon stimulation followed by ceramide generation through Mg(2+)-dependent sphingomyelinase induced delta PKC-specific translocation to the Golgi complex and that translocation results in delta PKC activation through tyrosine phosphorylation of the enzyme.     

Ceramide accumulation precedes caspase-3 activation during apoptosis of A549 human lung adenocarcinoma cells

Ceramide, the basic structural unit of sphingolipids, controls the balance between cell growth and death by inducing apoptosis. We have previously shown that accumulation of ceramide, triggered by hydrogen peroxide (H(2)O(2)) or by short-chain ceramide analogs, induces apoptosis of lung epithelial cells. Here we elucidate the link between caspase-3 activation, at the execution phase, and ceramide accumulation, at the commitment phase of apoptosis in A549 human lung adenocarcinoma cells. The induction of ceramide accumulation by various triggers of ceramide generation, such as H(2)O(2), C(6)-ceramide, or UDP-glucose-ceramide glucosyltransferase inhibitor dl-threo-1-phenyl-2-decanoylamino-3-morpholino-1-propanol, triggered the activation of caspase-3. This ceramide elevation also induced the cleavage of the death substrate poly(ADP-ribose) polymerase and was followed by apoptotic cell death. Ceramide-mediated apoptosis was blocked by a general caspase inhibitor, Boc-d-fluoromethylketone, and by overexpression of the antiapoptotic protein Bcl-2. Notably, overexpression of Bcl-2 reduced the basal cellular levels of ceramide and prevented the induction of ceramide generation by C(6)-ceramide, which implies ceramide generation as a possible target for the antiapoptotic effects of Bcl-2.     

Ceramide does not inhibit protein kinase C β-dependent phospholipase D activity stimulated by anti-Fas monoclonal antibody in A20 cells

We have investigated the roles of ceramide in Fas signalling leading to phospholipase D (PLD) activation in A20 cells. Upon stimulation of Fas signalling by anti-Fas monoclonal antibody, sphingomyelin hydrolysis and activation of PLD were induced. Also, the translocation of protein kinase C (PKC) βI and βII and the elevation of diacylglycerol (DAG) content were induced by Fas cross-linking. When phosphatidylcholine-specific phospholipase C (PC-PLC) was inhibited by D609, the Fas-induced changes in PLD activity, DAG content, and PKC translocation were inhibited. In contrast, D609 had no effect on Fas-induced alterations in sphingolipid metabolism, suggesting that changes in ceramide content do not account for Fas-induced PLD activation. Furthermore, C6-ceramide had no effect on Fas-induced PLD activation and PKC translocation. Taken together, these data might suggest that ceramide generated by Fas cross-linking does not affect PKC β-dependent PLD activity stimulated by anti-Fas monoclonal antibody in A20 cells.     

Dopamine release in PC12 cells is mediated by Ca(2+)-dependent production of ceramide via sphingomyelin pathway

A presynaptic membrane disturbance is an essential process for the release of various neurotransmitters. Ceramide, which is a tumor suppressive lipid, has been shown to act as a channel-forming molecule and serve as a precursor of ceramide-1-phosphate, which can disturb the cellular membrane. This study found that while permeable ceramide increases the rate of dopamine release in the presence of a Ca(2+)-ionophore, A23187, permeable ceramide-1-phosphate provoked its release even without the ionophore. The treatment of PC12 cells with the ionophore at concentrations < 2 microM produced ceramide via the sphingomyelin (SM) pathway with a concomitant release of dopamine, and no cell damage was observed. The addition of a Ca(2+) chelator, EGTA, to the medium inhibited the increase in the release of both the ceramide and dopamine. This suggests that ceramide might be produced by Ca(2+) and is implicated in the membrane disturbance associated with the release of dopamine as a result of its conversion to ceramide-1-phosphate. Consistent with these results, this study detected a membrane-associated and neutral pH optimum sphingomyelinase (SMase) whose activity was increased by Ca(2+). Together, these results demonstrate that ceramide can be produced via the activation of a neutral form of SMase through Ca(2+), and is involved in the dopamine release in concert with Ca(2+).     

The human T-cell leukemia virus type 1 p13II protein: effects on mitochondrial function and cell growth

p13(II) of human T-cell leukemia virus type 1 (HTLV-1) is an 87-amino-acid protein that is targeted to the inner mitochondrial membrane. p13(II) alters mitochondrial membrane permeability, producing a rapid, membrane potential-dependent influx of K(+). These changes result in increased mitochondrial matrix volume and fragmentation and may lead to depolarization and alterations in mitochondrial Ca(2+) uptake/retention capacity. At the cellular level, p13(II) has been found to interfere with cell proliferation and transformation and to promote apoptosis induced by ceramide and Fas ligand. Assays carried out in T cells (the major targets of HTLV-1 infection in vivo) demonstrate that p13(II)-mediated sensitization to Fas ligand-induced apoptosis can be blocked by an inhibitor of Ras farnesylation, thus implicating Ras signaling as a downstream target of p13(II) function.