Edelfosine is a prototypical member of the alkylphosphocholine class of antitumor drugs. Saccharomyces cerevisiae was used to screen for genes that modulate edelfosine cytotoxicity and identified sterol and sphingolipid pathways as relevant regulators. Edelfosine addition to yeast resulted in the selective partitioning of the essential plasma membrane protein Pma1p out of lipid rafts. Microscopic analysis revealed that Pma1p moved from the plasma membrane to intracellular punctate regions and finally localized to the vacuole. Consistent with altered sterol and sphingolipid synthesis resulting in increased edelfosine sensitivity, mislocalization of Pma1p was preceded by the movement of sterols out of the plasma membrane. Cells with enfeebled endocytosis and vacuolar protease activities prevented edelfosine-mediated (i) mobilization of sterols, (ii) loss of Pma1p from lipid rafts, and (iii) cell death. The activities of proteins and signaling processes are meaningfully altered by changes in lipid raft biophysical properties. This study points to a novel mode of action for an anti-cancer drug through modification of plasma membrane lipid composition resulting in the displacement of an essential protein from lipid rafts.The synthetic lipid edelfosine (also known as 1-O-octadecyl-2-Omethyl-rac-glycero-3-phosphocholine or ET-18-OCH3) is a prototypical member of the alkylphosphocholine class of cancer chemotherapeutic drugs. Alkylphosphocholines are effective drugs as they contain ether-linked fatty acids, as opposed to ester-linked fatty acids prevalent in endogenous phospholipids, and thus are much more resistant to cellular degradation by phospholipases (1-3). Because of their similarity in structure to phosphatidylcholine (PC), 5 the main experimentations to determine a mechanism of action for edelfosine and other alkylphosphocholines have focused on PC metabolism. This course of action was supported by observations that only alkylphospholipids with choline head groups, but not head groups found on other phospholipids such as ethanolamine or serine, were effective antitumor agents (1). Edelfosine and other choline-containing alkylphospholipids were found to inhibit PC synthesis and this correlated with inhibition of cell growth in various cancer cell lines (4 -7).Further metabolic labeling demonstrated that alkylphosphocholine drugs can inhibit the synthesis of PC-derived sphingomyelin, and this correlated with increased ceramide mass. Inhibition of de novo ceramide synthesis by the addition of the ceramide synthase inhibitor fumonisin B1 decreased ceramide levels and this was associated with increased resistance to alkylphosphocholines (8). As ceramide is a lipid second messenger whose accumulation can result in cytostasis or apoptosis (9, 10), an increase in cellular ceramide-mediated signaling was an alternate hypothesis proposed for alkylphosphocholine-mediated cell death (8).To gain further insight into the mechanism of action of alkylphosphocholine-mediated cytotoxicity we performed a genetic screen in Saccha...

The apoptotic program utilizes cellular membranes to transduce and generate operative signals. Lipids are major components of cellular membranes and have the potential to control the effectiveness of the signal by directing it to the proper location, being a source of new signals or as mediators in the response. These possible lipid functions are illustrated in the present review, focussing on the role that two different phospholipids, cardiolipin and phosphatidyl choline, play in apoptosis. Mitochondria have a central role in apoptosis, and many important aspects of the process mediated by this organelle converge through its distinctive lipid cardiolipin. Specifically, changes in cardiolipin metabolism have been detected in early steps of the death program and it is postulated (i) to mediate recruitment of pro apoptotic proteins like Bid to the mitochondria surface and (ii) to actively participate in the release of proteins relevant for the execution phase of apoptosis, like cytochrome c. Unlike the organelle specific distribution of cardiolipin, phosphatidylcholine is widely distributed among all organelles of the cell. The importance of phosphatidylcholine in apoptosis has been approached mainly through the study of the mode of action of (i) phosphatidylcholine anticancer analogues such as edelfosine and (ii) molecules that alter phosphatidylcholine metabolism, such as farnesol. The contribution of phosphatidylcholine metabolism to the apoptotic program is discussed, analyzing the experimental evidence available and pointing out some controversies in the proposed mechanisms of action.

Background:The antitumor lipid edelfosine kills yeast by inducing selective internalization of raft-associated proteins. Results: Impairing vesicular trafficking to the vacuole counteracted edelfosine-induced plasma membrane alterations without affecting internalization of the drug. Conclusion: Recycling of raft-associated proteins to the plasma membrane prevents edelfosine cytotoxicity. Significance: Vesicular trafficking is a critical process mediating edelfosine resistance in yeast that could be extrapolated to tumor cells.

In this study we demonstrate that the GAT1 and GAT2 genes encode the major glycerol-3-phosphate acyltransferase activities in Saccharomyces cerevisiae. Genetic inactivation of either GAT1 or GAT2 did not alter cell growth but inactivation of both resulted in growth cessation. Metabolic analyses of gat1 and gat2 yeast detected that the major differences were: (i) a 50% increase in the rate of triacylglycerol synthesis in gat1 yeast and a corresponding 50% decrease in gat2 yeast, and (ii) a 5-fold increase in glycerophosphocholine production through deacylation of phosphatidylcholine synthesized through the CDP-choline pathway in gat1 yeast, whereas gat2 yeast displayed a 10-fold decrease. To address why we observed alterations in phospholipid turnover specific to phosphatidylcholine produced through the CDP-choline pathway in gat1 and gat2 yeast we tested their sensitivity to various cytotoxic lysolipids and observed that gat2 cells were more sensitive to lysophosphatidylcholine, but not other lysolipids. To pursue the mechanism we analyzed their sensitivity to choline-containing lysolipids or drugs that could not be deacylated and/or reacylated. Our data showed that gat1 and gat2 yeast were resistant and sensitive to lysoplatelet activating factor, platelet activating factor, and the anti-tumor lipid edelfosine, respectively, indicating that their sensitivity to these compounds was not because of differences in rates of phosphatidylcholine deacylation. As growth of gat2 cells was impaired in the presence of ethanol, a phospholipase D (Spo14p) inhibitor, we inferred that phospholipase D may play important biologic and metabolic roles in phenotypes observed in gat yeast. Genetic inactivation of the SPO14 gene resulted in increased susceptibility, whereas expression of Escherichia coli diacylglycerol kinase relieved growth inhibition, to choline-containing lysolipids and drugs. Our results are consistent with a model whereby phosphatidic acid generated from phosphatidylcholine hydrolysis by Spo14p regulates susceptibility to choline-containing lysolipid analogs and drugs.