Apoptosis Triggered by 1-O-Octadecyl-2-O-methyl-rac-glycero-3-phosphocholine Is Prevented by Increased Expression of CTP:Phosphocholine Cytidylyltransferase

Abstract

A HeLa cell line was constructed for the regulation of CTP:phosphocholine cytidylyltransferase (CCT) expression via a tetracycline-responsive promoter to test the role of CCT in apoptosis triggered by exposure of cells to the antineoplastic phospholipid 1-O-octadecyl-2-Omethyl-rac-glycero-3-phosphocholine (ET-18-OCH 3 ). Basal CCT expression in the engineered HeLa cell line was the same as in control HeLa cells lines, and CCT activity and protein were elevated 25-fold following 48 h of induction with doxycycline. Increased CCT expression prevented ET-18-OCH 3 -induced apoptosis. Acylation of exogenous lysophosphatidylcholine circumvented the requirement for CCT activity by providing an alternate route to phosphatidylcholine, and heightened CCT expression and lysophosphatidylcholine supplementation were equally effective in reversing the cytotoxic effect of ET-18-OCH 3 . Neither CCT overexpression nor lysophosphatidylcholine supplementation allowed the HeLa cells to proliferate in the presence of ET-18-OCH 3 , indicating that the cytostatic property of ET-18-OCH 3 was independent of its effect on membrane phospholipid synthesis. These data provide compelling genetic evidence to support the conclusion that the interruption of phosphatidylcholine synthesis at the CCT step by ET-18-OCH 3 is the primary physiological imbalance that accounts for the cytotoxic action of the drug. ET-18-OCH 31 is a nonmetabolizable analog of LPC and belongs to the first generation of ether lipids tested as growth inhibitors (1). These compounds do not directly target DNA, and numerous studies have demonstrated a selective cytotoxic action of ET-18-OCH 3 against transformed cells in whole animals and tissue culture (2-8). Recent work has established that the cytotoxic effect of ET-18-OCH 3 is due to the ability of the antineoplastic phospholipids to induce apoptosis in sensitive cells (9 -12). A plethora of biological processes have been suggested as primary targets for the antineoplastic etherlinked phospholipids (for reviews, see Refs. 13-16). The long list of physiological imbalances includes the inhibition of phosphatidylinositol phospholipase C and calcium movements (17-19), protein kinase C-regulated functions (20 -24), lysophospholipid metabolism (25,26), and PtdCho synthesis (12,(27)(28)(29)(30)(31)(32)(33)(34)(35)(36)(37)(38)(39). Although some of the results are contradictory, it is clear that there are multiple targets for ET-18-OCH 3 , and it is not yet possible to distinguish the physiological imbalances that are causative from those that are either derivative or unrelated to the main event. Thus, a major contemporary focus in the field is to identify the critical cellular target(s) that are responsible for the cytotoxic and cytostatic actions of ET-18-OCH 3 .Our work has focused on the role of the inhibition of PtdCho synthesis in the mechanism of antineoplastic phospholipid action. PtdCho is essential for the survival of cultured cells because it is a major structural building block of biological membranes and the precurso...

Lipid metabolic changes caused by short-chain ceramides and the connection with apoptosis

“…Since C # -ceramide is converted into C # -SM by fibroblasts ( Figure 6 ; [12]), it seems plausible that the inhibition of PtdCho synthesis in BHK cells is caused by C # -SM synthesized from the added C # -ceramide. Such an interpretation is supported by the evidence that other artificial choline lipids such as hexadecylphosphocholine and 1-O-octadecyl-2-O-methylglycero-3-phosphocholine (' ET-18-OCH3 ') have an inhibitory effect on PtdCho synthesis through their action on cytidylyltransferase [25][26][27]. No increase in production of acylglycerols or phosphatidate has previously been reported using these inhibitors of cytidylyltransferase, but such accumulations would be logical consequences of this metabolic blockade.…”

“…Indeed, recent work shows that defective synthesis of PtdCho is by itself sufficient to cause apoptosis ; thus mutant Chinesehamster ovary (' CHO ') cells possessing a temperature-sensitive cytidylyltransferase undergo apoptosis when exposed to temperatures which inactivate this enzyme [28], and HeLa cells or HL60 cells are rescued from apoptosis caused by inhibitors of PtdCho synthesis if cytidylyltransferase is up-regulated [26] or if lysoPtdCho is added as an alternative source of PtdCho [27]. There is even a possibility that C # -ceramide as a natural metabolite could affect normal lipid synthesis, since at least in HL60 cells, endogenous C # -ceramide can be formed from platelet-activating factor [29].…”

Modulation of CTP:phosphocholine cytidylyltransferase by membrane curvature elastic stress

“…Hence, regulation of CTP:phosphocholine cytidylyltransferase (CCT) is critical for membrane biogenesis and for the production of new membrane during the S phase of the cell cycle (1,2). Furthermore, inhibition of CCT by using either biochemical (3)(4)(5)(6) or genetic (7) methods triggers programmed cell death. CCT activity is modulated by the association of its amphipathic helical domain with phospholipid bilayers (1,(8)(9)(10).…”

Role for phosphatidylinositol in nuclear envelope formation

“…To verify that the PI-PLC enzyme activity was promoting fusion, we added 19.6 µM ET-18-OCH $ , a specific inhibitor of PI-PLC with an IC &! of 0.4-9.6 µM [21]. We also added ET-18-OCH $ to the GTP-induced fusion assay mixture.…”