Arnold H. van der Luit

Ceramide: second messenger or modulator of membrane structure and dynamics?

The physiological role of ceramide formation in response to cell stimulation remains controversial. Here, we emphasize that ceramide is not a priori an apoptotic signalling molecule. Recent work points out that the conversion of sphingomyelin into ceramide can play a membrane structural (physical) role, with consequences for membrane microdomain function, membrane vesiculation, fusion/fission and vesicular trafficking. These processes contribute to cellular signalling. At the Golgi, ceramide takes part in a metabolic flux towards sphingomyelin, diacylglycerol and glycosphingolipids, which drives lipid raft formation and vesicular transport towards the plasma membrane. At the cell surface, receptor clustering in lipid rafts and the formation of endosomes can be facilitated by transient ceramide formation. Also, signalling towards mitochondria may involve glycosphingolipid-containing vesicles. Ceramide may affect the permeability of the mitochondrial outer membrane and the release of cytochrome c. In the effector phase of apoptosis, the breakdown of plasma membrane sphingomyelin to ceramide is a consequence of lipid scrambling, and may regulate apoptotic body formation. Thus ceramide formation serves many different functions at distinct locations in the cell. Given the limited capacity for spontaneous intracellular diffusion or membrane flip-flop of natural ceramide species, the topology and membrane sidedness of ceramide generation are crucial determinants of its impact on cell biology.

Alkyl-lysophospholipid Accumulates in Lipid Rafts and Induces Apoptosis via Raft-dependent Endocytosis and Inhibition of Phosphatidylcholine Synthesis

The synthetic alkyl-lysophospholipid (ALP), 1-O-octadecyl-2-O-methyl-rac-glycero-3-phosphocholine, is an antitumor agent that acts on cell membranes and can induce apoptosis. We investigated how ALP is taken up by cells, how it affectsde novo biosynthesis of phosphatidylcholine (PC), and how critical this is to initiate apoptosis. We compared an ALP-sensitive mouse lymphoma cell line, S49, with an ALP-resistant variant, S49AR. ALP inhibited PC synthesis at the CTP:phosphocholine cytidylyltransferase (CT) step in S49 cells, but not in S49AR cells. Exogenous lysophosphatidylcholine, providing cells with an alternative way (acylation) to generate PC, rescued cells from ALP-induced apoptosis, indicating that continuous rapid PC turnover is essential for cell survival. Apoptosis induced by other stimuli that do not target PC synthesis remained unaffected by lysophosphatidylcholine. Using monensin, low temperature and albumin back-extraction, we demonstrated that ALP is internalized by endocytosis, a process defective in S49AR cells. This defect neither involved clathrin-coated pit- nor fluid-phase endocytosis, but depended on lipid rafts, because disruption of these microdomains with methyl-β-cyclodextrin or filipin (sequestering cholesterol) or bacterial sphingomyelinase reduced uptake of ALP. Furthermore, ALP was found accumulated in isolated rafts and disruption of rafts also prevented the inhibition of PC synthesis and apoptosis induction in S49 cells. In summary, ALP is internalized by raft-dependent endocytosis to inhibit PC synthesis, which triggers apoptosis.

A new class of anticancer alkylphospholipids uses lipid rafts as membrane gateways to induce apoptosis in lymphoma cells

Single-chain alkylphospholipids, unlike conventional chemotherapeutic drugs, act on cell membranes to induce apoptosis in tumor cells. We tested four different alkylphospholipids, i.e., edelfosine, perifosine, erucylphosphocholine, and compound D-21805, as inducers of apoptosis in the mouse lymphoma cell line S49. We compared their mechanism of cellular entry and their potency to induce apoptosis through inhibition of de novo biosynthesis of phosphatidylcholine at the endoplasmic reticulum. Alkylphospholipid potency closely correlated with the degree of phosphatidylcholine synthesis inhibition in the order edelfosine > D-21805 > erucylphosphocholine > perifosine. In all cases, exogenous lysophosphatidylcholine, an alternative source for cellular phosphatidylcholine production, could partly rescue cells from alkylphospholipid-induced apoptosis, suggesting that phosphatidylcholine biosynthesis is a direct target for apoptosis induction. Cellular uptake of each alkylphospholipid was dependent on lipid rafts because pretreatment of cells with the raft-disrupting agents, methyl-β-cyclodextrin, filipin, or bacterial sphingomyelinase, reduced alkylphospholipid uptake and/or apoptosis induction and alleviated the inhibition of phosphatidylcholine synthesis. Uptake of all alkylphospholipids was inhibited by small interfering RNA (siRNA)–mediated blockage of sphingomyelin synthase (SMS1), which was previously shown to block raft-dependent endocytosis. Similar to edelfosine, perifosine accumulated in (isolated) lipid rafts independent on raft sphingomyelin content per se. However, perifosine was more susceptible than edelfosine to back-extraction by fatty acid-free serum albumin, suggesting a more peripheral location in the cell due to less effective internalization. Overall, our results suggest that lipid rafts are critical membrane portals for cellular entry of alkylphospholipids depending on SMS1 activity and, therefore, are potential targets for alkylphospholipid anticancer therapy. [Mol Cancer Ther 2007;6(8):2337–45]

Cell signalling: Control of free calcium in plant cell nuclei

Free calcium ions stimulate a huge variety of processes inside the cell, eliciting specific responses that depend on their spatio-temporal concentrations. Here we investigate how these changes in calcium concentration are triggered in the cytosol and nucleus of plant cells, and find that they are independently controlled in the two compartments. Our results indicate that in plants some processes in the nucleus may be executed in response to an autonomously regulated nuclear calcium signal, although it is not clear whether this happens in animal cells as well.

Resistance to alkyl-lysophospholipid-induced apoptosis due to downregulated sphingomyelin synthase 1 expression with consequent sphingomyelin- and cholesterol-deficiency in lipid rafts

The ALP (alkyl-lysophospholipid) edelfosine (1-O-octadecyl-2-O-methyl-rac-glycero-3-phosphocholine; Et-18-OCH3) induces apoptosis in S49 mouse lymphoma cells. To this end, ALP is internalized by lipid raft-dependent endocytosis and inhibits phosphatidylcholine synthesis. A variant cell-line, S49AR, which is resistant to ALP, was shown previously to be unable to internalize ALP via this lipid raft pathway. The reason for this uptake failure is not understood. In the present study, we show that S49AR cells are unable to synthesize SM (sphingomyelin) due to down-regulated SMS1 (SM synthase 1) expression. In parental S49 cells, resistance to ALP could be mimicked by small interfering RNA-induced SMS1 suppression, resulting in SM deficiency and blockage of raft-dependent internalization of ALP and induction of apoptosis. Similar results were obtained by treatment of the cells with myriocin/ISP-1, an inhibitor of general sphingolipid synthesis, or with U18666A, a cholesterol homoeostasis perturbing agent. U18666A is known to inhibit Niemann-Pick C1 protein-dependent vesicular transport of cholesterol from endosomal compartments to the trans-Golgi network and the plasma membrane. U18666A reduced cholesterol partitioning in detergent-resistant lipid rafts and inhibited SM synthesis in S49 cells, causing ALP resistance similar to that observed in S49AR cells. The results are explained by the strong physical interaction between (newly synthesized) SM and available cholesterol at the Golgi, where they facilitate lipid raft formation. We propose that ALP internalization by lipid-raft-dependent endocytosis represents the retrograde route of a constitutive SMS1- and lipid-raft-dependent membrane vesicular recycling process.

Different modes of internalization of apoptotic alkyl-lysophospholipid and cell-rescuing lysophosphatidylcholine

The synthetic alkyl-lysophospholipid (ALP), Et-18-OCH3 (1-O-octadecyl-2-O-methyl-rac-glycero-3-phosphocholine), can induce apoptosis in tumour cells. Unlike conventional chemotherapeutic drugs, ALP acts at the cell-membrane level. We have reported previously that ALP is internalized, and interferes with phosphatidylcholine (PC) biosynthesis de novo, which appeared to be essential for survival in lymphoma cells [Van der Luit, Budde, Ruurs, Verheij and Van Blitterswijk (2002) J. Biol. Chem. 277, 39541-39547]. Here, we report that, in HeLa cells, ALP accumulates in lipid rafts, and that internalization is inhibited by low temperature, monensin, disruption of lipid rafts and expression of a dominant-negative mutant of dynamin bearing a replacement of Lys44 with alanine (K44A). Thus ALP is internalized via raft- and dynamin-mediated endocytosis. Dynamin-K44A alleviated the ALP-induced inhibition of PC synthesis and rescued the cells from apoptosis induction. Additional cell rescue was attained by exogenous lysoPC, which after internalization serves as an alternative substrate for PC synthesis (through acylation). Unlike ALP, and despite the high structural similarity to ALP, lysoPC uptake did not occur via lipid rafts and did not depend on functional dynamin, indicating no involvement of endocytosis. Albumin back-extraction experiments suggested that (radiolabelled) lysoPC undergoes transbilayer movement (flipping). We conclude that ALP is internalized by endocytosis via lipid rafts to cause apoptosis, while exogenous cell-rescuing lysoPC traverses the plasma membrane outside rafts by flipping. Additionally, our data imply the importance of ether bonds in lyso-phospholipids, such as in ALP, for partitioning in lipid rafts.

Alkylphospholipids inhibit capillary-like endothelial tube formation in vitro: Antiangiogenic properties of a new class of antitumor agents

Synthetic alkylphospholipids (APLs), such as edelfosine, miltefosine and perifosine, constitute a new class of antineoplastic compounds with various clinical applications. Here we have evaluated the antiangiogenic properties of APLs. The sensitivity of three types of vascular endothelial cells (ECs) (bovine aortic ECs, human umbilical vein ECs and human microvascular ECs) to APL-induced apoptosis was dependent on the proliferative status of these cells and correlated with the cellular drug incorporation. Although confluent, nondividing ECs failed to undergo apoptosis, proliferating ECs showed a 3–4-fold higher uptake and significant levels of apoptosis after APL treatment. These findings raised the question of whether APLs interfere with new blood vessel formation. To test the antiangiogenic properties in vitro, we studied the effect of APLs using two different experimental models. The first one tested the ability of human microvascular ECs to invade a three-dimensional human fibrin matrix and form capillary-like tubular networks. In the second model, bovine aortic ECs were grown in a collagen gel sandwich to allow tube formation. We found that all three APLs interfered with endothelial tube formation in a dose-dependent manner, with a more than 50% reduction at 25 μmol/l. Interference with the angiogenic process represents a novel mode of action of APLs and might significantly contribute to the antitumor effect of these compounds.

Fas/CD95 down-regulation in lymphoma cells through acquired alkyllysophospholipid resistance: partial role of associated sphingomyelin deficiency.

The ALP (alkyl-lysophospholipid) edelfosine (1-O-octadecyl-2-O-methyl-rac-glycero-3-phosphocholine) induces apoptosis in S49 mouse lymphoma cells. A variant cell line, S49AR, made resistant to ALP, was found previously to be impaired in ALP uptake via lipid-raft-mediated endocytosis. In the present paper, we report that these cells display cross-resistance to Fas/CD95 ligation [FasL (Fas ligand)], and can be gradually resensitized by prolonged culturing in the absence of ALP. Fas and ALP activate distinct apoptotic pathways, since ALP-induced apoptosis was not abrogated by dominant-negative FADD (Fas-associated protein with death domain), cFLIP(L) [cellular FLICE (FADD-like interleukin 1beta-converting enzyme)-inhibitory protein long form] or the caspase 8 inhibitor Z-IETD-FMK (benzyloxycarbonyl-Ile-Glu-Thr-Asp-fluoromethylketone). ALP-resistant cells showed decreased Fas expression, at both the mRNA and protein levels, in a proteasome-dependent fashion. The proteasome inhibitor MG132 partially restored Fas expression and resensitized the cells to FasL, but not to ALP. Resistant cells completely lacked SM (sphingomyelin) synthesis, which seems to be a unique feature of the S49 cell system, having very low SM levels in parental cells. Lack of SM synthesis did not affect cell growth in serum-containing medium, but retarded growth under serum-free (SM-free) conditions. SM deficiency determined in part the resistance to ALP and FasL. Exogenous short-chain (C12-) SM partially restored cell-surface expression of Fas in lipid rafts and FasL sensitivity, but did not affect Fas mRNA levels or ALP sensitivity. We conclude that the acquired resistance of S49 cells to ALP is associated with down-regulated SM synthesis and Fas gene transcription and that SM in lipid rafts stabilizes Fas expression at the cell surface.

Phosphoinositide phosphatase SHIP-1 regulates apoptosis induced by edelfosine, Fas ligation and DNA damage in mouse lymphoma cells.

S49 mouse lymphoma cells undergo apoptosis in response to the ALP (alkyl-lysophospholipid) edelfosine (1-O-octadecyl-2-O-methyl-rac-glycero-3-phosphocholine), FasL (Fas ligand) and DNA damage. S49 cells made resistant to ALP (S49(AR)) are defective in sphingomyelin synthesis and ALP uptake, and also have acquired resistance to FasL and DNA damage. However, these cells can be re-sensitized following prolonged culturing in the absence of ALP. The resistant cells show sustained ERK (extracellular-signal-regulated kinase)/Akt activity, consistent with enhanced survival signalling. In search of a common mediator of the observed cross-resistance, we found that S49(AR) cells lacked the PtdIns(3,4,5)P(3) phosphatase SHIP-1 [SH2 (Src homology 2)-domain-containing inositol phosphatase 1], a known regulator of the Akt survival pathway. Re-sensitization of the S49(AR) cells restored SHIP-1 expression as well as phosphoinositide and sphingomyelin levels. Knockdown of SHIP-1 mimicked the S49(AR) phenotype in terms of apoptosis cross-resistance, sphingomyelin deficiency and altered phosphoinositide levels. Collectively, the results of the present study suggest that SHIP-1 collaborates with sphingomyelin synthase to regulate lymphoma cell death irrespective of the nature of the apoptotic stimulus.

Abstract 4025: SHIP-1 is involved in resistance to alkyl-lysophospholipid induced apoptosis in S49 T-cell lymphoma cells

Edelfosine (ALP, Et-18-OCH3) is a prototype alkyl-lysophospholipid that has effective antitumoral activity. It inserts in the membrane and accumulates in lipid rafts leading to apoptosis in S49 mouse T cell lymphoma cells. A variant cell line, made resistant to ALP (S49AR) shows in impaired uptake of ALP. S49AR cells are not only resistant to a variety of ALP analogues, but also to DNA damage and Fas/CD95 death receptor induced apoptosis, suggesting another cause for resistance in addition to impaired ALP uptake. Here we studied the ALP resistance of S49AR. We observe an upregulation of phosho-PKB/Akt and phospho-ERK1/2 which are key proteins in the canonical survival signaling pathways. However inhibition of ERK and/or PKB/Akt using pharmacological inhibitors has no effect on the resistance of the S49AR cells to apoptotic stimuli. PKB/Akt and ERK1/2 activation is regulated by PI(3,4,5)P3 formation. The levels of phosphoinositides in the various cell lines were measured with HPLC. The cell lines resistant to apoptotic inducing agents show decreased levels of the diverse phosphopoinositides. SHIP-1 is a SH2 domain containing inositol phosphatase that removes the 5′ phosphate of PI(3,4,5)P3 and is mainly expressed in hemotopoietic cells. It decreases the pool of PI(3,4,5)P3 thereby negatively regulating the activation of PKB/Akt. We see down regulation of SHIP-1 both on gene expression and protein level in the S49AR compared to S49 cells. Other components of the phosphoinositide metabolism, like PI3Kinase and SHIP2, did not show any changes. Resensitization of the S49AR cells to ALP leads to restoration of SHIP-1 expression. Knocking down SHIP-1 using siRNA causes resistance to various ALPs, DNA damage and Fas/CD95 death receptor induced apoptosis. Microarray analysis show that the S49AR and S49siSHIP cells show remarkably similar expression profile which are distinct from the expression profiles shown by S49 and S49mock cells. Concluding, SHIP-1 is downregulated in ALP resistant T cell lymphoma cells concurrent with a general downregulation of phosphoinositide levels in the cells which is associated with the resistance to ALP induced apoptosis. How ALP resistance is caused is yet unknown and is the scope of current research. Note: This abstract was not presented at the AACR 101st Annual Meeting 2010 because the presenter was unable to attend. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 4025.