Pharmacological Inhibition of Phosphatidylcholine Biosynthesis Is

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[CANCER RESEARCH 60, 5204–5213, September 15, 2000] Pharmacological Inhibition of Phosphatidylcholine Biosynthesis Is Associated with Induction of Phosphatidylinositol Accumulation and Cytolysis of Neoplastic Cell Lines

Robert E. Finney,1 Edward Nudelman, Thayer White, Stuart Bursten, Peter Klein, Laura L. Leer, Nu Wang, David Waggoner, Jack W. Singer, and Robert A. Lewis Cell Therapeutics, Inc., Seattle, Washington 98119

ABSTRACT action of PC-specific PLD (PC-PLD; Fig. 1), and generation of PA by PC-PLD has been associated with neoplastic transformation. In par- De novo production of phosphatidic acid (PA) in tumor cells is required ticular, PC-PLD activity was augmented in surgically resected human for phospholipid biosynthesis and growth of tumor cells. In addition, PA gastric carcinoma cells and human breast cancer cells, as compared production by phospholipase D has been cited among the effects of certain oncogenes and growth factors. In this report, it has been demonstrated with adjacent noncancerous tissues (6, 7), and this enzymatic activity that enhanced phospholipid metabolism through PA in tumor cells can be was increased in response to each of a broad class of mitogenic exploited pharmacologically for development of anticancer agents, such as stimulants, including platelet-derived growth factor (8, 9), epidermal CT-2584, a cancer chemotherapeutic drug candidate currently in Phase II growth factor (10), basic fibroblast growth factor (11), insulin (12, clinical trials. By inhibiting CTP:choline-phosphate cytidylyltransferase 13), and phorbol esters (14). Mutations in growth factors or growth (CT), CT-2584 caused de novo phospholipid biosynthesis via PA to be factor receptors for some of these activators of PC-PLD elicited the shunted away from phosphatidylcholine (PC) and into phosphatidylinosi- unregulated activities of oncogenes (e.g., v-sis, erb-B, and HER2/ tol (PI), the latter of which was doubled in a variety of CT-2584-treated neu). Furthermore, elevation of PA levels was observed in fibroblasts tumor cell lines. In contrast, cytotoxic concentrations of cisplatin did not neoplastically transformed with ras and fps oncogenes (15), and an induce accumulation of PI, indicating that PI elevation by CT-2584 was increase in PC-PLD activity was correlated with expression of the src not a general consequence of chemotherapy-induced cell death. Consistent with this mechanism of action, propranolol, an inhibitor of PA phospho- oncogene (16). PA has been shown recently to have targets implicated hydrolase and phosphatidylcholine biosynthesis, was also cytotoxic to in neoplastic transformation and growth factor signal transduction tumor cell lines, induced PI accumulation, and potentiated the activity of pathways. These targets include phospholipase C␥ (9, 17), Ras-Gap CT-2584 in cytotoxicity assays. As expected from biophysical properties of (18–20), Raf-1 (21, 22), and a protein tyrosine phosphatase (23). anionic phospholipids on cellular membranes, CT-2584 cytotoxicity was Additionally, some responses connected with the neoplastic pheno- associated with disruption and swelling of endoplasmic reticulum and type may also be mediated by utilization of PA to produce other mitochondria. We conclude that CT-2584 effects a novel mechanism of intracellular signaling molecules, including DAG and the potent mi- cytotoxicity to cancer cells, involving a specific modulation of phospho- togen, LPA (Fig. 1). Thus, in tumor cells, critical trafficking of lipid metabolism. phospholipid metabolism through PA is a result not only of de novo biosynthesis but also of extracellular and oncogene-induced process- INTRODUCTION ing of other cellular lipids. In this report, we demonstrate that CT-2584, an anticancer agent 2 Although PA is not a major constituent of biological membranes, currently in Phase II clinical trials, exploits the enhanced trafficking of it is a key intermediate in phospholipid biosynthesis. De novo bio- lipids through PA in tumor cells to selectively alter the phospholipid synthesis of PA from glycerol via glycerol phosphate and then LPA is composition of these cells and to produce a resultant cytotoxicity. accomplished by condensation of that moiety with a saturated or monounsturated fatty acyl-CoA by the action of a LPA acyl transfer- MATERIALS AND METHODS ase (Fig. 1; Ref. 1). The newly synthesized PA is a precursor for biosynthesis of major constituents of biological membranes that in- Chemicals. PBS, growth medium, and all cell culture reagents were pur- clude PC, PE, and PS through DAG as an intermediate, or for chased from Life Technologies, Inc. (Gaithersburg, MD). All phospholipids synthesis of anionic phospholipids including PI and cardiolipin that were used as standards were purchased from Avanti, Inc. (Alabaster, AL). HP-TLC-HP-HLF silica gel TLC plates were purchased from Analtech, Inc. through CDP-DAG as an intermediate. This large-scale biosynthesis (Newark, DE). [U-14C]glycerol and [oleoyl-1-14C]DAG were purchased from of phospholipids is required for cell proliferation, especially for that in Amersham Life Science (Elk Grove, IL). 5-[3H]Cytidine and [methyl-14C]cho- tumor cells. In particular, PC biosynthesis has been implicated in line were purchased from American Radiolabeled Chemicals, Inc. (St. Louis, control of cell proliferation (2, 3), and inhibition of PC biosynthesis in MO). DL-Propranolol and cisplatin were purchased from Sigma Chemical Co. tumor cell lines has been associated with apoptosis of tumor cells (St. Louis, MO). (4, 5). Cell Culture. MCF-7 (human breast adenocarcinoma cells), NCI-H460 Smaller quantities of PA can also be generated from PC by the (human large cell lung carcinoma cells), and NCI-H23 (human non-small cell lung carcinoma cells) were obtained from the American Type Culture Collec- tion (ATCC). Human bone marrow stromal (HBMS) cells were prepared from Received 11/22/99; accepted 7/28/00. The costs of publication of this article were defrayed in part by the payment of page normal human bone marrow transplant donors under IRB-approved protocols charges. This article must therefore be hereby marked advertisement in accordance with (24). MCF-7, NCI-H460, and NCI-H23 cells were grown in RPMI supple- 18 U.S.C. Section 1734 solely to indicate this fact. mented with 10% FBS (heat inactivated at 56°C for 30 min), 100 units/ml 1 To whom requests for reprints should be addressed, at Cell Therapeutics, Inc., 201 penicillin, 100 ␮g/ml streptomycin, and 2 mML-glutamine. HBMS cells were Elliott Avenue West, Suite 400, Seattle, WA 98119. Phone: (206) 282-7100: Fax: (206) 284-6206; E-mail: [email protected]. grown in McCoy’s 5A medium supplemented with 12.5% heat inactivated 2 The abbreviations used are: PA, phosphatidic acid; LPA, lysophosphatidic acid; PC, FBS, 12.5% heat inactivated horse serum, 100 units/ml penicillin, 100 ␮g/ml phosphatidylcholine; PE, phosphatidylethanolamine; PS, phosphatidylserine; DAG, di- streptomycin, 2 mML-glutamine, 10ng/ml basic fibroblast growth factor, 1% acylglycerol; PI, phosphatidylinositol; CDP, cytidine diphosphate diacylglycerol; PLD, vitamins, 0.4% essential amino acids, 0.04 mM nonessential amino acids, 1 mM phospholipase D; CFU-GM, colony forming unit granulocyte-macrophage; MBM, murine ␮ bone marrow; C-M, chloroform:methanol; LPP, lipid phosphate phosphatase; CDS, CDP- sodium pyruvate, 0.075% (w/v) sodium bicarbonate, and 0.36 g/ml hydro- DAG synthase; CT, cytidylyltransferase. cortisone (25). All incubations were at 37°C under 5% CO2, 95% air. 5204

MBM cells were extruded from femurs with RPMI 1640 containing 10% FBS, using a 20-gauge needle and a 1-ml syringe. The MBM cells were adjusted to a concentration of 1.0 ϫ 106 cells/ml and incubated with CT-2584 for 6 or 8 h at concentrations ranging from 5 to 30 ␮M. After incubation, the cells were pelleted at 1000 ϫ g for 5 min, washed twice, and counted. Mononuclear cell counts were performed in 2% glacial acetic acid. MBM cells were added to a standard CFU-GM assay (Stem Cell Technologies, Inc., Vancouver, BC) in quadruplicate at 50,000 cells/plate and incubated for 7 days. Spleen condi- tioned medium (2% final volume) was used as the source of colony stimulating factor. Colonies (Ͼ40 cells) were counted and averaged among quadruplicate samples. Average colony counts in treated samples were normalized for comparisons between experiments. Linear curve-fitting software (Delta Graph version 3.5.2) was used to plot relative colony counts as a function of CT-2584 Fig. 1. Biochemical pathways regulating the trafficking of phospholipids through PA. concentration. An IC50 was interpolated across multiple points between 0.0 GK, glycerol kinase; GPAT, glycerophosphate acyltransferase; LPAAT, lisophosphatidic and 1.0. acid acyltransferase; LPP, lipid phosphate phosphatase; DK, diacylglycerol kinase; PIS,PI synthase; CK, choline kinase; CT, CTP:choline-phosphate cytidyltransferase; CPT, CDP- Phospholipid Extraction and Partition. Tumor cell lines and HBMS cells choline:1,2-DAG choline phosphotransferase. were grown to 70–90% confluency. Incubations with [14C]glycerol (0.04– 0.08 ␮Ci/ml), [14C]-DAG (0.08 ␮Ci/ml), and [14C]choline (0.16 ␮Ci/ml) were carried out either prior to, concurrent with, or after addition of CT-2584 to the Chemical Synthesis of CT-2584. 11-Bromo-1-undecene was prepared cell cultures for the defined periods stated in “Results.” Subsequent to treat- from ␻-undecylenyl alcohol (Aldrich) by treatment with phosphorus tribro- ment with test agents, the medium was aspirated, and the cells were washed mide (0.4 mole equivalents) and pyridine (0.2 mole equivalents) in toluene (as once with ice-cold PBS and were scraped into cold PBS with a rubber solvent) at 4°C, followed by heating to 50°C for an overnight period. Treat- policeman. The harvested cells and cellular fragments were then pelleted at ment of 11-bromo-1-undecene with 3-chloroperoxybenzoic acid (1.05 mole 1500 rpm in a Beckman CS-6 centrifuge (GH 3.8 swinging bucket rotor) over equivalents) in 1,2-dichloroethane, initially at 10°C, with subsequent warming 5 min. After the supernatant was removed, 6 ml of chloroform-methanol (C-M) to room temperature, provided 11-bromo-1,2-epoxyundecane. Sodium theo- (2:1, v/v) were added to the cell pellets. The organic cell suspension was bromine was obtained after treating a solution of theobromine in ethanol with vortexed for 3 min, followed by bath sonication for 10 min with occasional

sodium methoxide (1.0 mole equivalent) followed by solvent evaporation. vortexing. One ml of 0.2 M KCl/0.2 M H3PO4 was added to the organic Heating an equimolar mixture of 11-bromo-1, 2-epoxyundecane and sodium sonicates. The contents were capped and vortexed for 3 min and then bath theobromine in DMSO at 80°C provided 1-(10,11-epoxyundecyl)-3,7-dimeth- sonicated for 5 min. To obtain phase separation, the tubes were centrifuged at ylxanthine, which, upon further heating with dodecylamine (2 mole equiva- 1000 ϫ g for 10 min. In each tube, a white band of solid material was evident lents) in DMSO to 100°C, yielded 1-(11-dodecylamino-10-hydroxyundecyl)- at the interface between the upper and lower phases. The aqueous upper phase 3,7-dimethylxanthine (CT-2584). CT-2584 purity was determined to be 96– was aspirated and discarded. A Pasteur pipette was then inserted past the 99% by 1H NMR, 13C NMR and HPLC analysis. Treatment of CT-2584 with margin of the solid band into the lower phase to effect a quantitative recovery methanesulfonic acid (0.95 mole equivalents) in hot isopropanol, followed by of the uncontaminated lower phase by aspiration. The lower phase from each addition of heptane, gave the methanesulfonic acid salt of CT-2584 which was tube was evaporated to dryness in a 37°C nitrogen needle evaporator (N-evap), dissolved in a solution of ethanol, deionized water and Tween 80 (70:29.5:0.5) with the addition of ethanol to azeotropically remove water. The dried lipid and stored as a 10 mM stock solution at Ϫ20°C. material was redissolved by vortexing in 0.5 ml C-M (2:1), dried again, and ␮ Cytotoxicity Assays. The LC50 of CT-2584 was evaluated on a panel of 35 dissolved once more in 100 l of C-M (2:1) with brief vortexing and sonica- human tumor cell lines. Sub-confluent cultures grown in medium per Ameri- tion. can Type Culture Collection or supplier were treated for 24 h with 1–50 ␮M Multi-One Dimensional TLC and Quantitation of Lipids. Methods used CT-2584 for 24 h. The cells were then incubated for an additional 24 h in for separating lipids using multi-one dimensional TLC were modified from growth medium lacking drug before determining their viability. Adherent cells those described by White et al. (26). Twenty-␮l portions of extracted lipids were assayed using BCECF, a mixture of 2Ј,7Ј-bis-(2-carboxyethyl)-5- were spotted 7 cm from the bottom of TLC plates in 0.5-cm lanes, each carboxyfluorescein and 2Ј,7Ј-bis-(2-carboxyethyl)-6-carboxyfluorescein ace- separated by 0.7 cm. The lipids were initially resolved using a mobile phase of toxymethyl esters per the manufacturer’s instructions (Molecular Probes, Inc., chloroform:methanol:ammonium hydroxide (65:30:4) for 90 min. This al- Eugene, OR). For nonadherent cells, CT96 AQ (Promega, Inc., Madison, WI) lowed for maximal separation of PC from PI and other polar phospholipids. To

was added directly to the cells in growth medium (20 ␮l of dye/100 ␮lof detect PC (approximate Rf, 0.15) and other mobile phospholipids, the top half medium). After 1–4 h of incubation, cell viability was evaluated by measuring of the plate was sprayed with a 0.05% solution of the lipid-associating dye, absorbance at 490 nm on a model EL 340 plate reader (Bio Tek Instruments, primulin, and the positions of all bands of stained lipid were visualized using Inc., Winooski, VT). a handheld UV lamp, UVL-21 (UVP, Inc., San Gabriel, CA). The plate was Additional cell viability assays on MCF-7, NCI-H460, NCI-H23, and then scored horizontally with a diamond pen and broken just below the PC HBMS were performed. Subconfluent cultures were incubated with specified band. The resultant lower portion of the plate was dried in an air stream and concentrations of CT-2584 or vehicle for the indicated times, and viability was rotated 180° for the next chromatographic step in chloroform:methanol:acetic determined using 10% Alamar Blue per the manufacturer’s instructions (Trek acid (glacial):water (70:30:15:6, v/v), which was allowed to advance two- Diagnostics Systems, Inc., Chicago, IL). Reduced Alamar Blue (a measure of thirds the distance to the top of the plate. The plate was dried again and cell viability) was measured at an excitation wavelength of 530 nm for chromatographed in chloroform:methanol:acetic acid:water (85:12.5:12.5:3, emission at 590 nm using a model Cytofluor 2300/2350 fluorescence plate v/v), which was allowed to ascend to the top of the plate. After the plate was reader (Millipore, Inc., Bedford, MA). After subtracting background fluores- dried once more, the final resolution of the lipids was achieved after repeating cence, the percentage of viability was determined by dividing fluorescence of this latter chromatography procedure, with solvent exposure halted 5 min after test samples by fluorescence of vehicle-treated cells. The concentration of it had reached the top of the plate. After drying, each plate was sprayed with

agent causing 50% lethality was defined as the LC50. In some experiments, cell primulin and scanned by laser-excited fluorescent detection on a STORM 840 death was also determined using Sytox-Green, as described by the manufac- imaging system (Molecular Dynamics, Sunnyvale, CA). The quantity of sep- turer (Molecular Probes, Inc., Eugene, OR). Cell death is associated with a arated phospholipids on the TLC plates (in nmol) was determined by integra- compromised cell membrane, leading to an association of Sytox-Green with tion of variable pixel intensities on Imagequant software and extrapolation cellular nucleic acids and, thus, enhanced fluorescence. For this assay, the from standard curves. After analysis of lipid mass by primulin, the plates were excitation wavelength was 485 nm for an emission at 530 nm. exposed to STORM Phosphor Screens for 96 h, and the amount of [14C]glyc- CFU-GM Assay. To determine whether CT-2584 was cytotoxic to murine erol in separated lipids was quantitated with Imagequant. Unless otherwise bone marrow (MBM) progenitor cells, a CFU-GM assay was performed. stated, the amount of 14C associated with individual phospholipids (in pixel 5205

using PA:PC vesicles at Kms for CTP and PA, as described (30). PC-PLD activity was measured in a broken cell assay as described (31) and in intact CT-2584-treated cells using butan-1-ol trapping (32). DAG kinase activity was evaluated using an N-octyl-␤-D-glucopyranoside-containing assay, with 1,2- dioleoyl-sn-glycerol as the substrate, as described (33). PI synthase activity

was assayed at Kms for CDP-DAG and inositol in a detergent-free assay, as described (34). Fig. 2. Chemical structure of CT-2584, 1(11-dodecylamino-10-hydroxyundecyl)-3,7- Electron Microscopy. NCI-H460 cells were plated into 60-mm culture dimethylxanthine. dishes at a density of 176 cells/mm2 and incubated overnight. After treatment with 10 ␮M CT-2584 or vehicle, the cells were trypsinized and pelleted at 1000 rpm in a Beckman CS-6 centrifuge (GH 3.8 swinging bucket rotor) for 2 min units) was normalized to 14C associated with total phospholipids (PC, PE, PI, at 4°C. The cell pellet was washed once with 3 ml of ice-cold PBS and once PS, and PA). with 3 ml of cold 0.2 M cacodylate solution. The final cell pellet was fixed Identity and purity of each separated lipid was determined by comigration overnight at 4°C in half-strength Karnovsky’s fixative and postfixed in 1% with standards and confirmed by electrospray ionization mass spectrometry. collidine-buffered osmium tetroxide. After dehydration, cells were embedded The analyzed lipids were quantitatively recovered in single bands and were in Epon 812. Ultrathin sections were stained using saturating aqueous uranyl homogeneous, based upon these analyses. acetate and lead tartrate and examined using a JOEL 100 SX transmission Analysis of CK, CT, and CPT Activities in Cell-free Extracts. Subcon- electron microscope operating at 80 kV. Electron microscopy was performed fluent MCF-7 cells were incubated with vehicle (70% ethanol and 0.5% Tween by the electron microscopy facility at the Fred Hutchinson Cancer Research 80) or 10 ␮M CT-2584 for 2 h and then rinsed and harvested in an ice-cold 50 Center (Seattle, WA). mM Tris-HCl (pH 7.5) buffer containing 50 mM NaCl, 1 mM each DTT, EDTA, EGTA, and benzamidine, and 1 ␮g/ml each pepstatin A, soybean trypsin RESULTS inhibitor, E64C, and Pefabloc, with or without added CT-2584 (10 ␮M). The suspended cells were then sonicated with a Branson Sonifer 450 microtip at Cytotoxic Activity of CT-2584. Because sphingolipids such as power setting 1, 99% duty for three times 30 s on ice. [14C]Choline (1.6 ␮Ci, ceramide, sphingosine, and sphingosine 1-phosphate are cytotoxic to ␮ ␮ ␮ 40 nmol), ATP (5 mol), CTP (1 mol), and MgCl2 (5 mol) were added to tumor cell lines in concert with their modulation of phospholipid 2 mg of cellular material in a final incubation volume of 500 ␮l. After a 30 min metabolism, a series of lipid-like compounds with various degrees of incubation at 37°C, the incubation was quenched by the addition of chloro- similarities to sphingolipids was synthesized. Included were com- form:methanol (1:1, v/v) and extracted by the method of Bligh and Dyer (27). pounds with amino alcohol and dimethylxanthine domains. CT-2584 Aliquots of the aqueous and organic phases were separated on silica thin layer (Fig. 2) was selected from this compound library for development and plates using methanol:2.4% NaCl:H O:NH OH (50:12.5:37.5:5) or CHCl : 2 4 3 is currently in Phase II clinical trials as a cancer chemotherapeutic ethanol:AA:H2O (75:45:12:6), respectively. Radiolabeled materials in the extracts were identified by comigration with known standards, and radioactivity drug candidate. was quantitated using the STORM 840 imaging system (Molecular Dynamics, Twenty-four h of continuous exposure to CT-2584 was cytotoxic to Sunnyvale, CA) and Imagequant software. 35 human tumor cell lines, including those resistant to multiple drugs, 3 Ϯ ␮ Ϯ [ H]Cytidine Incorporation into CDP Diacylglycerol. MCF-7 cells were with LC50sof4.0 2.3 M (mean SD). The panel of tumor cell plated into 150 mm culture dishes containing 25 ml of growth medium at a lines tested included breast, lung, prostate, sarcoma, colon, melanoma, density of 1.5 ϫ 106 cells/dish. After 48 h incubation, (at ϳ70–80% conflu- brain, ovarian, and leukemia cells. Nontumorigenic HBMS cells were ency), the medium was decreased to 20 ml and supplemented with either 10 Ϯ 3–4-fold less sensitive to CT-2584 cytotoxicity (LC 50, 15.7 3.1 ␮M CT-2584 or vehicle for 2 h. [3H]Cytidine (100 ␮Ci) was then added and the ␮M) than the panel of human tumor cell lines. Similarly, the colony- cultures were incubated for an additional 2 h. After washing twice with forming capacity of murine bone marrow progenitor cells (CFU-GM) ice-cold PBS, cells were harvested in cold PBS containing a protease inhibitor was 3–4-fold less sensitive to inhibition by CT-2584 (IC , 13.9 Ϯ 6.1 cocktail (1 mM benzamidine and 1 mg/ml each Pefabloc, E64, leupeptin, 50 ␮ pepstatin A, and soybean trypsin inhibitor). Phospholipids were then extracted M) than were human tumor cell lines to CT-2584-mediated cytotox- according to the method of Bligh and Dyer (27), except that 2 M KCl icity.

containing 0.2 M H3PO4 was used, instead of water, to separate phases. Lipids CT-2584 Decreases PC and Increases PI Accumulation in Tu- in the organic extract were separated using multi-one dimensional TLC, as mor Cell Lines. Cytotoxic concentrations of CT-2584 were evalu- described above, except that the first dimension was run twice in chloroform: ated for effects on phospholipid metabolism in MCF-7 and NCI-H460 methanol:ammonia:water (60:35:4:1), and the second dimension was run once cells. [14C]Glycerol was allowed to approach a steady state in the in chloroform:methanol:acetic acid:water (50:28:4:8). Separated lipids were cellular phospholipid pools by incubating for 24 h. The tumor cell visualized using primulin and scanned with laser-excited fluorescent detection lines were then treated with specified concentrations of CT-2584 or on a STORM 840 imaging system. The area comigrating with CDP-DAG vehicle, in the continued presence of [14C]glycerol, for the indicated standards was then scraped, and 3H beta emissions were quantitated by scintillation counting. time, and lipids were extracted and separated using multi-one dimen- Evaluation of LPP, CDS, PC-PLD, DAG Kinase, and PI Synthase sional TLC. TLC plates were sprayed with primulin to stain the Activities. Crude membranes, isolated by sonication and 100,000 ϫ g cen- separated phospholipids. The relative quantities of the separated phos- trifugation of ECV304 cells (a T24 variant) overexpressing human LPP-1, pholipids were determined by measuring both beta emissions from the LLP-2, LLP-3, or CDP-DAG synthase-1, were used as the source of the stated [14C]radionuclide and the mass by the amount of primulin stain enzyme activities. PC-PLD activity was partially purified from membranes of associated with each phospholipid. SF-9 insect gut epithelial cells overexpressing the enzyme. Cytosol from Jurkat [14C]PC decreased and [14C]PI increased in both NCI-H460 cells cells, which contains mostly type I DAG kinase (28), was used as a source of (Fig. 3, a and b) and MCF-7 cells (Fig. 3d) at cytotoxic concentrations this enzyme activity, and membranes from MCF-7 cells were used in PI of CT-2584. A concentration-dependent relationship was observed synthase assays. All enzyme assays were performed such that no more than 5% within the tested range, with effects observed at or above 2–3 ␮M of the substrates was consumed during the reaction to insure linearity of the ␮ 14 reaction kinetics. Protein fractions were preincubated for 15–30 min at 25°C or CT-2584. At 15 M CT-2584, the amount of [ C]PC relative to total 37°C in appropriate buffer containing protease inhibitors and 0–40 ␮M CT- phospholipid (PE, PC, PI, PS, and PA) was reduced by 14–15%, and 14 2584. LPP activity was assayed using 100 ␮M 1,2-dioleoyl-[33P]-sn-glycero- the relative amount of [ C]PI was increased by 8–15%, as compared 14 14 phosphate in Triton micelles or in PC vesicles in the presence of 5 mM with levels observed in vehicle-treated cells. [ C]PE, [ C]PS, and N-ethylmaleimide, as described (29). CDP-DAG synthase activity was assayed [14C]PA did not markedly change over the concentrations tested. The 5206

Fig. 3. Concentration-dependent effect of CT-2584 on phospholipid metabolism in NCI-H460 and MCF-7 cells. NCI-H460 cells (a–c) and MCF-7 cells (d and e) were incubated with [14C]glycerol for 24 h and then with specified concentrations of CT-2584 or vehicle in the continued presence of [14C]glycerol for 4 h. Lipids were extracted, separated by multi-one dimensional TLC, and stained with primulin. Both the amount of 14C associated with phospholipids (a for NCI-H460 cells) and the amount of primulin stain associated with phospholipids were quantitated. Data are presented as the percentage of 14C associated with each lipid entity relative to 14C associated with the sum of the indicated phospholipids (PE, PC, PI, PS and PA; b and d)or as the percentage of primulin stain associated with each lipid entity relative to the sum of the indicated phospholipids (c and e). The average of triplicate determinations (b–e) are represented. Bars, SD.

decrease in [14C]PC represented 24–25% of the PC measured in To evaluate phospholipid metabolism, phospholipids in NCI-H460 vehicle-treated cells, whereas the increase in [14C]PI was 124–170% and MCF-7 cells were labeled with [14C]gylcerol for 24 h. The cells over that in vehicle-treated cells. Primulin staining of phospholipids were then treated with either 10 ␮M CT-2584 or vehicle for specified separated by TLC indicated a comparable decrease in mass of PC, times, in the continued presence of [14C]glycerol, and phospholipids representing 18–20% of PC in vehicle-treated cells, and increase in were extracted and evaluated. A decrease in [14C]PC and a corre- the mass of PI, which was 71–125% over that in vehicle-treated cells sponding increase in [14C]PI were observed within2hoftreatment (Fig. 3, c and e). Similar decreases in PC and increases in PI were with CT-2584 in both NCI-H460 cells (Fig. 4b) and MCF-7 cells (Fig. demonstrated in NCI-H23 human non-small cell lung cancer cells and 4c). Maximal changes were observed by 8 h, at which time the amount DU-145 human prostate cancer cells (data not shown). of [14C]PC relative to total phospholipid was reduced by 10–17%, and CT-2584 Induces PI Accumulation Prior to Cell Death. Some [14C]PI was increased 9–13%, as compared with levels observed in changes in phospholipids would be expected to occur after cells have vehicle-treated cells. The decrease in [14C]PC represented 16–28% of died, because of loss of regulation and compartmentalization of lipid- PC measured in vehicle-treated cells, and the increase in [14C]PI was metabolizing enzymes, and would not have a causal association with 102–127% over that in vehicle-treated cells. A small change in cytotoxicity. In contrast, changes in phospholipid composition prior to [14C]PE was observed by8hinMCF-7 cells only, whereas little or no overt signs of cell death would be compatible with a cause-effect change in [14C]PS or [14C]PA was seen in either cell line. Primulin relationship. To test whether changes in PC and PI occurred as a result staining of phospholipids separated by TLC indicated a roughly of cell death or whether these changes occurred prior to cell death, comparable decrease in the mass of PC, representing 15–20% of PC time courses for cytolysis of NCI-H460 and MCF-7 cells were deter- in vehicle-treated cells, and increase in mass of PI, which was 81– mined and compared with the time courses for changes in phospho- 96% over that in vehicle-treated cells (data not shown). Therefore, lipid metabolism. When treated with 10 ␮M CT-2584, NCI-H460 CT-2584 induced quantitative changes in PC and PI mass prior to the Ϯ ␮ Ϯ ␮ (LC50, 3.3 0.3 M) and MCF-7 (LC50, 4.5 0.5 M) cells earliest demonstration of cell death. remained viable for more than 4 h, as indicated by exclusion of Induced PI Accumulation Is Specific to CT-2584. To test Sytox-Green, which is a measure of plasma membrane integrity (Fig. whether accumulation of PI is specific to CT-2584 treatment or 4a). Some cell death was detectable at 6 h, and all cells were killed whether other chemotherapeutic agents might induce the same re- within 24 h of exposure to the compound. Similar time courses of cell sponse as a nonspecific event associated with cytotoxicity, the effect death were obtained using the Alamar Blue viability assay (data not of CT-2584 and cisplatin on phospholipids in NCI-H23 (non-small shown). Although death by apoptosis was not evident in NCI-H460 or cell lung carcinoma) cells was examined. NCI-H23 cells were selected

MCF-7 cells, cytotoxicity in other cell lines such as U937 (monocytic based upon their sensitivity to both CT-2584 and cisplatin. The LC50 leukemia) was associated with apoptosis, as evidenced by a decrease for treatment with CT-2584 was 4 ␮M, and 8 ␮M approached the ␮ in DNA content and by analysis of DNA fragmentation (data not LC100 (Fig. 5a). The LC50 for cisplatin was 15 M, and although an ␮ shown). LC100 was not achieved at concentrations up to 100 M, the cytotoxic 5207

PLD. However, in both cellular-based assays and cell-free assays, CT-2584 had no effect on PC-PLD activity (data not shown). Alter- natively, the data are consistent with inhibition of de novo PC biosynthesis with concurrent induction of de novo PI biosynthesis. To evaluate the effects of CT-2584 on de novo PC and PI biosynthesis, short-term labeling experiments were performed using [14C]glycerol, 14 14 [ C]DAG, and [ C]choline. MCF-7 cells were treated with 10 ␮M CT-2584 or vehicle for 2 h. [14C]Gylcerol, [14C]DAG, or [14C]choline was then incubated with cells for 2–4 additional h in the continued presence of CT-2584 or vehicle. Phospholipids were evaluated as described previously, except the [14C]choline incorporation into PC was normalized to total phospholipid mass because choline does not incorporate into PE, PS, PA, or PI. Pulse-labeled PC was reduced by 82% using [14C]glycerol (Fig. 6a), by 59% using [14C]DAG (Fig. 6b), and by 95% using [14C] choline (Fig. 6c). Pulse-labeled PI was increased by 550% using [14C]glycerol and by 560% using [14C]DAG. As opposed to charged lipids such as phospholipids, DAG is neutral, and its uptake into mammalian cells has been reported by others (35) and confirmed for MCF-7 cells (data not shown). Therefore, CT-2584 inhibits PC biosynthesis and induces PI biosynthesis. Inhibition of PC biosynthesis appears to be distal to DAG biosynthesis, because DAG incorporation into PC was reduced with CT-2584. In addition, because [14C]PI derived from the [14C]DAG precursor increased in CT-2584-treated cells, it appears that CT-2584 is capable of diverting DAG away from PC biosynthesis and into PI biosynthesis. Effect of CT-2584 on Cell-free Choline Kinase, CT, and CPT Activity. The final biosynthetic step for formation of PC is a condensation reaction with DAG and CDP-choline (Fig. 1). Because CT-2584 inhibition of PC biosynthesis appeared to occur distal to DAG production, the effects of CT-2584 on key enzymes involved in production of CDP-choline (referred to as Kennedy pathway enzymes; CK, CT, and CPT; Ref. 36) were evaluated. MCF-7 cells were treated with 10 ␮M CT-2584 or vehicle for 2 h, and crude cell lysates were prepared in the continued presence of 10 ␮M CT-2584 or vehicle. The cell lysates were then incubated with [14C]choline for 30 min at 37°C, and incorporation into phosphocholine, CDP-choline, and PC was evaluated (Fig. 7). [14C]Phosphocholine was not decreased in lysates of CT-2584-treated cells as compared with those from vehicle-treated cells, indicating that CK was not a target for CT-2584. A major effect of CT-2584 was found on production of [14C]CDP-phosphocholine, which was decreased by 79% in lysates of Fig. 4. Time course for cytotoxicity and accumulation of PI in response to CT-2584. NCI-H460 cells (a, f) and MCF-7 cells (a, Ⅺ) were treated with 10 ␮M CT-2584 or CT-2584-treated cells, as compared with those of vehicle-treated vehicle for the indicated times and assayed for cytotoxicity using Sytox-Green reagent. cells. Although a very limited amount of [14C]PC was detected from ϳ Data are presented as the percentage of maximal fluorescence, which was 5-fold over lysates of treated or untreated cells, the degree of inhibition with background fluorescence. For phospholipid analysis, MCF-7 cells (b) and NCI-H460 cells 14 (c) were incubated with [ C]glycerol for 24 h and then with 10 ␮M CT-2584 or vehicle CT-2584 was similar to that observed for inhibition of CDP-DAG for the specified times in the continued presence of [14C]glycerol. Lipids were extracted production, suggesting that CT-2584 did not have a major effect on and analyzed as in Fig. 3. CPT activity. CT-2584-induced PI Production Is Associated with Augmented effect at 37.5 ␮M was almost maximal at ϳ80% of the cells (Fig. 5b). Production of CDP-DAG. The accumulation of PI at the expense of As had been shown for MCF-7 and NCI-H460 cells, NCI-H23 cells PC, as induced by CT-2584, could occur either by augmented traf- treated with 4 ␮M CT-2584 for8hor10␮M CT-2584 (2.5 times the ficking of phospholipids through PA and CDP-DAG intermediates, by 14 14 LC50)for4hor8hdecreased [ C]PC and accumulated [ C]PI (Fig. decreased utilization of PI to form PI-polyphosphates, or by decreased 5c). No changes were observed for [14C]PE, [14C]PS, or [14C]PA. In transfer and exchange of PI and PC catalyzed by the PI transfer contrast, there were no changes in [14C]PC, [14C]PI, or any of the protein (37, 38). To determine whether trafficking of lipids through other phospholipids after treatment with 15 ␮M cisplatin for8hor CDP-DAG was increased after treatment with CT-2584, MCF-7 cells ␮ ␮ 3 37.5 M (2.5 times the LC50) cisplatin for4hor8h(Fig. 5d). were treated with either 10 M CT-2584 or vehicle for 2 h. [ H]Cy- CT-2584 Inhibits de Novo PC Biosynthesis and Induces de Novo tidine was then added, and the cultures were incubated for an addi- PI Biosynthesis. That the decrease in PC mass and the increase in PI tional2hinthecontinuous presence of CT-2584 or vehicle. mass are approximately equal in CT-2584-treated cells (see Fig. 3) Incorporation of [3H]cytidine into CDP-DAG, as quantitated after suggests that PI is accumulating at the expense of PC. One possibility TLC, increased from 11,354 Ϯ 591 dpm/mg total cell protein in to account for this observation is that PC serves as a precursor for PI vehicle-treated cells to 45,979 Ϯ 2,636 dpm/mg total cell protein in production after treatment with CT-2584 through activation of PC- CT-2584-treated cells. Because the total amount of cell-associated 5208

Fig. 5. Effects of CT-2584 and cisplatin on viability and phospholipid metabolism in NCI-H23, human non-small cell lung carcinoma cells. NCI- H23 cells were treated with the indicated concentrations of CT-2584 (a) or cisplatin (b) for 24 h and assayed for viability 24 h later using Alamar Blue. Data are presented as a percentage of vehicle- treated cells. Arrows, concentrations of each drug used for analyses of phospholipid metabolism. For phospholipid analyses, NCI-H23 cells were incubated with [14C]glycerol for 24 h and then with indicated concentrations of CT-2584 (c) or cisplatin (d) in the continued presence of [14C]glycerol for the indicated times (4 or 8 h). Vehicle control represents cells treated with the same solublizing vehicle as used for CT-2584 or cisplatin. Phospho- lipids were extracted and analyzed as in Fig. 3. Bars, SD.

radioactivity in CT-2584-treated cells was severalfold less than in CT-2584 (compare Figs. 3 and 4 to Fig. 8a). It was, therefore, not vehicle-treated cells (data not shown), the increase in [3H]cytidine- surprising to find that propranolol, like CT-2584, was cytotoxic to labeled CDP-DAG could not be attributable to an increased rate of MCF-7 cells at concentrations that induced accumulation of PI. incorporation of cytidine into cells. Instead, these data would suggest The combined effects of propranolol and CT-2584 on cytotoxicity that the 3–4-fold increase in [3H]cytidine incorporation in CT-2584- were evaluated on MCF-7 cells. The cells were pretreated with the treated cells is likely an underestimation of the increased production indicated concentrations of propranolol or vehicle for 1 h. CT-2584 or of CDP-DAG. In contrast to this analysis of [3H]cytidine-labeled vehicle was then added to cultures for an additional 10 h. The medium cells, cell-free assays of CDP-DAG synthase activity were unaffected containing drugs was then aspirated from the cell cultures and re- by CT-2584 (data not shown), suggesting that this enzyme was not a placed with fresh growth medium lacking drugs for 14 h. Viability direct target for the drug. In a separate cell-free assay for PI synthase was then determined using the Alamar Blue assay and plotted as a activity, CT-2584 was also without effect (data not shown). percentage of propranolol-treated (plus vehicle of CT-2584) cells Effect of Propranolol on Phospholipid Metabolism and CT- (Fig. 8b). The combined effect of CT-2584 and propranolol on cyto- 2584 Cytotoxicity. If augmented production of PA in tumor cells toxicity was greater than the sum of effects attributable to the indi- causes them to be more sensitive to CT-2584 than are nontumorigenic vidual drugs. Therefore, concentrations of propranolol that induced PI cells, then pharmacological agents that further enhance trafficking of accumulation also potentiated CT-2584 cytotoxicity (compare Fig. 8, phospholipids into PA might also be expected to increase production compare a with b). Because only the S- enantiomer of propranolol is of PI and enhance CT-2584 cytotoxicity. Independent of its ␤-adre- a ␤-adrenergic receptor antagonist and both the R- and S- enantiomers nergic antagonist capacity, propranolol has been shown to inhibit both of propranolol synergized with CT-2584 in cytotoxicity assays (data N-ethylmaleimide-sensitive and -insensitive forms of LPP, a class of not shown), the effect of propranolol on phospholipid metabolism was enzymes that catalyze the hydrolytic release of the phosphate group therefore unrelated to cyclic 3Ј,5Ј-AMP production. from PA with the resultant formation of DAG (see Fig. 1; Ref. 39). Effects of CT-2584 and Propranolol on PI Biosynthesis Are Therefore, propranolol would be expected to enhance trafficking of Additive. If cytotoxicity of CT-2584 and propranolol depend upon PA into metabolites other than DAG, including PI. MCF-7 cells reduced PC or induced PI production in tumor cell lines, then the labeled with [14C]glycerol for 24 h were then treated with specified potentiation of CT-2584 cytotoxicity by propranolol would be ex- concentrations of propranolol or vehicle, in the continued presence of pected to be reflected by either an additive or synergistic effect on PI [14C]glycerol, for 5 h. 14C-Labeled phospholipids were extracted and accumulation. To test this possibility, the phospholipid pool in MCF-7 analyzed (Fig. 8a). Propranolol induced accumulation of [14C]PI in cells was radiolabeled with [14C]glycerol for 24 h. Propranolol, at 100 14 MCF-7 cells and caused a corresponding decrease in [ C]PC. Meas- ␮M final concentration, was then added to cultures. After 1-h incuba- 14 urable increases in [ C]PI were induced with propranolol concentration with propranolol, 10 ␮M CT-2584 or vehicle was added to the cell 14 14 tions as low as 25 ␮M, without producing effects on [ C]PE, [ C]PS, cultures, and the phospholipids were extracted 4 h later (Fig. 8c). Both or [14C]PA. Similar changes were observed by analysis of primulin- CT-2584 and propranolol decreased [14C]PC and increased [14C]PI in stained phospholipids (data not shown). The effects of propranolol on the tumor cell line, and a combination of both agents had an additive phospholipid metabolism were therefore similar to those induced by effect on changes in [14C]PC and [14C]PI. Because propanolol evoked 5209

LPP-3 (data not shown). Furthermore, CT-2584 had no direct effect on type I DAG kinase (data not shown). The Cytotoxic Effect of CT-2584 Is Associated with Swelling and Disruption of Mitochondria and the Rough Endoplasmic Reticulum. Accumulation of anionic phospholipids is expected to cause loss of membrane integrity attributable to decreased lamellar tendency, change or loss of function of intrinsic membrane proteins, or increased likelihood of intracellular membrane fusion, especially in

Fig. 6. Effect of CT-2584 on de novo PC and PI biosynthesis in MCF-7 cells. MCF-7 cells were treated with 10 ␮M CT-2584 or vehicle for 2 h. Cells were then incubated with [14C]glycerol (a), [14C]DAG (b), or [14C]choline (c) in the continued presence of CT-2584 for 2 additional h. Phospholipids were extracted and analyzed as in Fig. 3, except [14C]choline incorporation into PC was normalized to total phospholipid mass (PE, PE, PI, PS, and PA), as determined by primulin stain. Bars, SD.

Fig. 8. Concentration-dependent effects of propranolol on phospholipid metabolism and CT-2584 cytotoxicity in MCF-7 cells. a, to determine effects on phospholipid metabolism, MCF-7 cells were incubated with [14C]glycerol for 24 h and then with the indicated concentrations of propranolol or vehicle for 5 h. Phospholipids were then extracted and analyzed as in Fig. 3. Concentrations of propranolol that are reproducibly found to have some cytotoxicity are indicated. b, potentiation of CT-2584 cytotoxicity by Fig. 7. Effect of CT-2584 on cell-free [14C]choline incorporation into phosphocholine, propranolol. MCF-7 cells were treated with specified concentrations of propranolol for 1 h, followed by the addition of the indicated concentrations of CT-2584 for 10 h. Cells CDP-choline, and PC. MCF-7 cells were treated with 10 ␮M CT-2584 (f) or vehicle (Ⅺ) were then washed free of drugs and incubated an additional 14 h in growth medium for 2 h. Cell lysates were then prepared either in the continued presence of 10 ␮M CT-2584 or vehicle. [14C]Choline incorporation into phosphocholine, CDP-choline, and PC was lacking drugs, and viability was determined using Alamar Blue. Data are presented as a determined and represented as a percentage of total 14C label in the assay. Bars, SD. percentage of viability of propranolol-treated (plus vehicle for CT-2584) cells. Viabilities of cells treated with propranolol (and vehicle of CT-2584) in this particular experiment were as follows: 96% viable at 25 ␮M, 87% viable at 50 ␮M, 86% viable at 100 ␮M, and 66% viable at 200 ␮M propranolol. c, effects of a combination of CT-2584 and propranolol phospholipid changes that are similar to those induced by CT-2584, on phospholipid metabolism. MCF-7 cells were incubated with [14C]glycerol for 24 h. 14 i.e., decreased PC and augmented PI production, it was questioned Treatments, in the continued presence of [ C]glycerol, included 4 h with 10 ␮M CT-2584, 5 h with 100 ␮M propranolol, or 5 h with 100 ␮M propranolol in combination with 10 ␮M whether CT-2584 might also inhibit LPP. This activity was, however, CT-2584 for the final 4 h. Lipids were extracted and analyzed as described in Fig. 3. unaffected by CT-2584 in separate assays of LPP-1, LPP-2, and Similar effects were observed in multiple experiments. Bars, SD. 5210

Fig. 9. Electron micrographs of CT-2584-treated NCI-H460 cells. NCI-H460 cells were treated with vehicle (a)or10␮M CT-2584 (b and c) for 10 h, harvested, and prepared for transmission electron microscopy at the indicated magnifications. PM, plasma membrane; NM, nuclear membrane; M, mitochondrion; rER, rough endoplasmic reticulum.

organelles rich in calcium. To determine the effect of CT-2584 on and PI biosynthesis do not occur in mitochondria, where cardiolipin is membrane integrity and subcellular structure, NCI-H460 cells were synthesized, or that overproduced cardiolipin is rapidly degraded. treated with 10 ␮M CT-2584 or vehicle for periods of from 1 to 10 h, Although we have not definitively demonstrated an etiological role and changes in subcellular morphology were evaluated by electron of the reduced PC and enhanced PI production by CT-2584 in the microscopy. The most striking morphological effects of CT-2584 death of tumor cells, the accumulated data are thus far consistent with were marked swelling and loss of structural integrity of the rough this hypothesis. Notably, inhibition of PC biosynthesis and accumu- endoplasmic reticulum and mitochondria as early as 5 h, as depicted lation of PI occurred at concentrations of CT-2584 that were cytotoxic for a 10-h exposure in Fig. 9. In contrast, even at the 10-h time point, (Fig. 3) but prior to biochemical evidence of cytotoxicity (Fig. 4). the plasma membrane and nuclear membranes of CT-2584-treated Cytotoxic concentrations of propranolol, a drug with an enzymatic cells were morphologically unaltered from their appearance in cells effect in the same biochemical pathway, also reduced PC and aug- treated with the vehicle for CT-2584. mented PI production in tumor cell lines. In addition, the effects of CT-2584 and propranolol on PC and PI were additive, and concen- DISCUSSION trations of propranolol that induced PI accumulation potentiated CT- 2584 cytotoxicity (Fig. 8). Finally, the effect of CT-2584 on PI was CT-2584 was cytotoxic in vitro for a broad panel of tumor cell not demonstrable at cytotoxic concentrations of an agent (cisplatin) lines, including those resistant to multiple drugs. In an independent that killed the same cell type (NCI-H23) by a different, defined study, CT-2584 was also shown to produce tumor growth delay in mechanism (Fig. 6). human hepatocellular carcinoma cells in severe combined immunod- Quantitatively, PC decreased by as much as 25%, and PI doubled eficient mice. Equivalent antitumor effects were seen with hepatocel- with the final mole percentage of PI approaching 20–25% of total luar carcinoma cells that were resistant to multiple drugs on the basis major phospholipids (Figs. 3 and 4) in CT-2584-treated cells. Thus, of overexpression of mdr1 (40). Furthermore, Phase I clinical data the balance between primarily structural phospholipids (PC and PE) suggested antitumor activity against a variety of cancers and indicated and the anionic phospholipid, PI, is dramatically affected by CT-2584. 3 that plasma concentrations up to 3–4 ␮M were repeatedly achieved, The structure, function, and integrity of biological membranes are which is consistent with concentrations presently shown to elicit primarily dependent upon lipid composition. PC and the sphingolipid, biochemical events in tumor cells in vitro. sphingomyelin, appear to play structural roles in stabilizing bilayer CT-2584 inhibited PC biosynthesis, as measured by incorporation (lamellar) structure and extended, high-radius shapes (e.g., plasma of [14C]glycerol, [14C]DAG, and [14C]choline, and induced PI biomembranes), whereas PE promotes the formation of unilamellar in- synthesis, as measured by incorporation of [14C]glycerol and verted phases and membranes with small radii of curvature (e.g., [14C]DAG, in tumor cell lines (Fig. 6). Inhibition of CTP:choline- Golgi bodies and exocytotic vesicles; Refs. 41–44). In contrast, ani- phosphate CT was determined to be responsible for the effects of onic phospholipids such as PA, PI, and cardiolipin (Fig. 1) are CT-2584 on PC biosynthesis (Fig. 7). That [3H]cytidine labeling of primarily required for the function of intrinsic membrane proteins in CDP-DAG in cellular assays increased with CT-2584 treatment sug- various organelles (45, 46) and for membrane fusion (47). Therefore, gests that the increased production of PI used CDP-DAG and, by the lipid composition of cellular organelles must be balanced to inference, PA intermediates. Moreover, because utilization of maintain structural integrity while, at the same time, accommodating [14C]DAG for PI biosynthesis was augmented in CT-2584-treated proteins in functional states and regulating membrane fusion. If bal- cells (Fig. 6), it appears that inhibition of CT may divert DAG away ance in lipid composition of membranes is not maintained, then the from PC biosynthesis and into PI biosynthesis. That the enzyme structural integrity and function of organelles and cells would be activities that mediate phospholipid metabolism between DAG and PI compromised. (CDS, PI synthase, LPP, and DAG kinase) were unaffected by CT- On the basis of the biophysical data obtained from model systems 2584 (data not shown) is consistent with this interpretation. Somewhat and biological systems (48), both the massive decrease in PC and surprisingly, the CT-2584-effected enhancement of phospholipid traf- increase in PI as produced by CT-2584 would be expected to con- ficking through PA and CDP-DAG did not augment production of tribute to a loss of lamellar structure and membrane integrity. This cardiolipin (data not shown). This suggests either that PI biosynthesis would be attributable to decreased capacity to maintain a lipid bilayer, is favored under the experimental conditions, that the effects on PC change or loss of function in intrinsic membrane proteins, or increased likelihood of intracellular membrane fusion (41, 42, 47–50). The 3 Unpublished observations. effect of PI would be most dysregulatory in organelles rich in calcium, 5211

(e.g., the endoplasmic reticulum and the mitochondrion), because the REFERENCES 2ϩ spherical radii of the PI polar head groups, when aggregated by Ca , 1. West, J., Tompkins, C. K., Balantac, N., Nudelman, E., Meengs, B., White, T., would be too small, relative to the spherical radii of the average acyl Bursten, S., Coleman, J., Kumar, A., Singer, J. W., and Leung, D. W. Cloning and domains, to maintain ordered geometry. As a result, the presence of expression of two human lysophosphatidic acid acyltransferase cDNAs that enhance cytokine-induced signaling responses in cells. DNA Cell Biol., 16: 691–701, 1997. 2ϩ Ca would be expected to further decrease the lamellar tendencies of 2. Jackowski, S. Coordination of membrane phospholipid synthesis with the cell cycle. the bilayer, leading to formation of intramembrane micellar subre- J. Biol. Chem., 269: 3858–3867, 1994. 3. Jackowski, S. Cell cycle regulation of membrane phospholipid metabolism. J. Biol. gions and loss of membrane integrity (41, 42, 48–50). Consistent with Chem., 271: 20219–20222, 1996. this interpretation, decreased production of PC and increased produc- 4. Cui, Z., Houweling, M., Chen, M. H., Record, M., Chap, H., Vance, D. E., and Terce, F. A genetic defect in phosphatidylcholine biosynthesis triggers apoptosis in Chinese tion of PI was associated with disruption of membrane integrity of hamster ovary cells. J. Biol. Chem., 271: 14668–14671, 1996. both mitochondria and endoplasmic reticulum (Fig. 9). 5. Baburina, I., and Jackowski, S. Apoptosis triggered by 1-O-octadecyl-2-O-methyl- The attraction of the biochemical approach exemplified by CT- rac-glycero-3-phosphocholine is prevented by increased expression of CTP: phosphocholine cytidylyltransferase. J. Biol. Chem., 273: 2169–2173, 1998. 2584 is theoretically appealing. PC biosynthesis through Kennedy 6. Uchida, N., Okamura, S., and Kuwano, H. Phospholipase D activity in human gastric pathway intermediates has been associated with proliferation of tumor carcinoma. Anticancer Res., 19: 671–675, 1999. 7. Uchida, N., Okamura, S., Nagamachi, Y., and Yamashita, S. Increased phospholipase cell lines (2, 3), and PC turnover was augmented in Ras-transformed D activity in human breast cancer. J. Cancer Res. Clin. Oncol., 123: 280–285, 1997. cells (51). The association of augmented production of Kennedy 8. Plevin, R., Cook, S. J., Palmer, S., and Wakelam, M. J. Multiple sources of sn-1,2- pathway intermediates is further exemplified by the observations that diacylglycerol in platelet-derived-growth-factor-stimulated Swiss 3T3 fibroblasts. Evidence for activation of phosphoinositidase C and phosphatidylcholine-specific oncogenes (ras, src, raf, and mos) induced choline kinase activity in phospholipase D. Biochem. J., 279: 559–565, 1991. rodent cells, that generation of phosphocholine is essential for the 9. Lee, Y. H., Kim, H. S., Pai, J. K., Ryu, S. H., and Suh, P. G. Activation of phospholipase D induced by platelet-derived growth factor is dependent upon the mitogenic action of certain growth factors (52–54), and that at least level of phospholipase C-␥ 1. J. Biol. Chem., 269: 26842–26847, 1994. some human tumors contain augmented levels of phosphomonoesters 10. Yeo, E. J., and Exton, J. H. Stimulation of phospholipase D by epidermal growth that include phosphocholine and phosphoethanolamine (55–59). In factor requires protein kinase C activation in Swiss 3T3 cells. J. Biol. Chem., 270: 3980–3988, 1995. addition, intrinsic oncogenic mechanisms (e.g., the presence of onco- 11. Motoike, T., Bieger, S., Wiegandt, H., and Unsicker, K. Induction of phosphatidic genes derived from receptor and non-receptor tyrosine kinases and acid by fibroblast growth factor in cultured baby hamster kidney fibroblasts. FEBS Lett., 332: 164–168, 1993. members of the ras gene family) would also be expected to induce PA 12. Farese, R. V., Konda, T. S., Davis, J. S., Standaert, M. L., Pollet, R. J., and Cooper, production in tumor cells by activation of PC-PLD (6–16). By inhib- D. R. Insulin rapidly increases diacylglycerol by activating de novo phosphatidic acid synthesis. Science (Washington DC), 236: 586–589, 1987. iting CT and PC biosynthesis, such high rates of PA production in 13. Donchenko, V., Zannetti, A., and Baldini, P. M. Insulin-stimulated hydrolysis of tumor cells, either through high rates of phospholipid biosynthesis or phosphatidylcholine by phospholipase C and phospholipase D in cultured rat hepa- turnover (e.g., through PC-PLD), would be expected to yield higher tocytes. Biochim. Biophys. Acta, 1222: 492–500, 1994. 14. Mizunuma, M., Tanaka, S., Kudo, R., and Kanoh, H. Phospholipase D activity of levels of PI biosynthesis in tumor cells than in non-tumor cells after human amnion cells stimulated with phorbol ester and bradykinin. Biochim. Biophys. CT-2584 treatment. Hence, tumor cells would be expected to be more Acta, 1168: 213–219, 1993. 15. Martin, A., Duffy, P. A., Liossis, C., Gomez-Munoz, A., O’Brien, L., Stone, J. C., and sensitive to cytotoxic mechanisms evoked by CT-2584 than would Brindley, D. N. Increased concentrations of phosphatidate, diacylglycerol and cer- non-tumor cells. And finally, because CT-2584 resides in cellular amide in ras- and tyrosine kinase (fps)-transformed fibroblasts. Oncogene, 14: 1571– membranes (data not shown) and does not have to traverse cellular 1580, 1997. 16. Song, J., and Foster, D. A. v-Src activates a unique phospholipase D activity that can membranes to reach its biological target, it is not likely to be subject be distinguished from the phospholipase D activity activated by phorbol esters. to the drug-extruding molecular pumps that are overexpressed in Biochem. J., 294: 711–717, 1993. 17. Jones, G. A., and Carpenter, G. The regulation of phospholipase C-␥ 1 by phospha- multidrug resistance tumor cells (60, 61). Hence, it was not surprising tidic acid. Assessment of kinetic parameters. J. Biol. Chem., 268: 20845–20850, that CT-2584 was active against multidrug-resistant tumor cell lines 1993. 18. Tsai, M. H., Roudebush, M., Dobrowolski, S., Yu, C. L., Gibbs, J. B., and Stacey, both in cell culture (as reported herein) and in vivo (40). D. W. Ras GTPase-activating protein physically associates with mitogenically active Agents, such as etoposide, camptothecin, farnesol, and cheleryth- phospholipids. Mol. Cell Biol., 11: 2785–2793, 1991. rine (62) and ether lipids such as 1-O-octadecyl-2-O-methyl-rac- 19. Tsai, M. H., Hall, A., and Stacey, D. W. Inhibition by phospholipids of the interaction between R-ras, rho, and their GTPase-activating proteins. Mol. Cell Biol., 9: 5260– glycero-3-phosphocholine (5) have also been shown to inhibit PC 5264, 1989. biosynthesis and induce apoptosis, although the effects of these agents 20. Tsai, M. H., Yu, C. L., Wei, F. S., and Stacey, D. W. The effect of GTPase activating protein upon Ras is inhibited by mitogenically responsive lipids. Science (Washing- on PI biosynthesis have not been described. At least for etopside and ton DC), 243: 522–526, 1988. camptothecin, it is not clear whether effects on PC biosynthesis 21. Ghosh, S., Strum, J. C., Sciorra, V. A., Daniel, L., and Bell, R. M. Raf-1 kinase significantly contribute to the cytotoxic activities of the agents, be- possesses distinct binding domains for phosphatidylserine and phosphatidic acid. Phosphatidic acid regulates the translocation of Raf-1 in 12-O-tetradecanoylphorbol- cause these agents are thought to function primarily by inhibition of 13-acetate-stimulated Madin-Darby canine kidney cells. J. Biol. Chem., 271: 8472– topoisomerases (63). Although ether lipids appear to inhibit both 8480, 1996. 22. Rizzo, M. A., Shome, K., Vasudevan, C., Stolz, D. B., Sung, T. C., Frohman, M. A., protein kinase C as well as PC biosynthesis, ectopic expression of Watkins, S. C., and Romero, G. Phospholipase D and its product, phosphatidic acid, CTP:phosphocholine cytidyltransferase was capable of abrogating mediate agonist-dependent raf-1 translocation to the plasma membrane and the apoptosis induced by 1-O-octadecyl-2-O-methyl-rac-glycero-3-phos- activation of the mitogen-activated protein kinase pathway. J. Biol. Chem., 274: 1131–1139, 1999. phocholine (5), suggesting that PC biosynthesis was central to its 23. Chalfant, C. E., Kishikawa, K., Mumby, M. C., Kamibayashi, C., Bielawska, A., and cytotoxic mechanism of action. Hannun, Y. A. Long chain ceramides activate protein phosphatase-1 and protein phosphatase-2A. Activation is stereospecific and regulated by phosphatidic acid. We conclude that phospholipid metabolism in tumor cells may J. Biol. Chem., 274: 20313–20317, 1999. represent an opportunity for development of anticancer agents, as 24. Nemunaitis, J., Ruillian, M., Tompkins, C., Andrews, D. F., and Singer, J. W. Stromal cells, growth factors and matrix proteins. In: N. C. Gorin and L. Douay (eds.), exemplified by CT-2584. Because CT-2584 possesses a unique mech- Experimental Hematology Today, pp. 53–57. New York: Springer-Verlag, 1989. anism of action involving reduced PC mass and production of the 25. Sensebe, L., Li, J., Lilly, M., Crittenden, C., Herve, P., Charbord, P., and Singer, J. W. anionic phospholipid PI, because it is efficacious against tumor cell Nontransformed colony-derived stromal cell lines from normal human marrows. I. Growth requirement and myelopoiesis supportive ability. Exp. Hematol., 23: 507– lines resistant to multiple drugs, and because the drug is well tolerated 513, 1995. by patients, we propose that CT-2584 be used as an index compound 26. White, T., Bursten, S., Federighi, D., Lewis, R. A., and Nudelman, E. High-resolution separation and quantification of neutral lipid and phospholipid species in mammalian for using the altered phospholipid dynamics of neoplastic cells to cells and sera by multi-one-dimensional thin-layer chromatography. Anal. Biochem., target cancer chemotherapy. 258: 109–117, 1998. 5212

27. Bligh, E. G., and Dyer, W. J. A rapid method of total lipid extraction and purification. 46. Fry, M., and Green, D. E. Cardiolipin requirement for electron transfer in complex Can. J. Biochem. Physiol., 37: 911–917, 1959. I and III of the mitochondrial respiratory chain. J. Biol. Chem., 256: 1874–1880, 28. Yamada, K., Sakane, F., and Kanoh, H. Immunoquantitation of 80 kDa diacylglycerol 1981. kinase in pig and human lymphocytes and several other cells. FEBS Lett., 244: 47. Zimmerberg, J., Vogel, S. S., and Chernomordik, L. V. Mechanisms of membrane 402–406, 1989. fusion. Annu. Rev. Biophys. Biomol. Struct., 22: 433–466, 1993. 29. Truett, A. P., Bocckino, S. B., and Murray, J. J. Regulation of phosphatidic acid 48. Aguilar, L., Ortega-Pierres, G., Campos, B., Fonseca, R., Ibanez, M., Wong, C., phosphohydrolase activity during stimulation of human polymorphonuclear leuko- Farfan, N., Naciff, J. M., Kaetzel, M. A., Dedman, J. R., and Baeza, I. Phospholipid cytes. FASEB J., 6: 2720–2725, 1992. membranes form specific nonbilayer molecular arrangements that are antigenic. 30. Carman, G. M., and Kelley, M. J. CDPdiacylglycerol synthase from yeast. Methods J. Biol. Chem., 274: 25193–25196, 1999. Enzymol., 209: 242–247, 1992. 31. Brown, H. A., Gutowski, S., Moomaw, C. R., Slaughter, C., and Sternweis, P. C. 49. Kinnunen, P. K. On the principles of functional ordering in biological membranes. ADP-ribosylation factor, a small GTP-dependent regulatory protein, stimulates phos- Chem. Phys. Lipids, 57: 375–399, 1991. pholipase D activity. Cell, 75: 1137–1144, 1993. 50. Marsh, D. Lipid-protein interactions and heterogeneous lipid distribution in mem- 32. Exton, J. H. Signaling through phosphatidylcholine breakdown. J. Biol. Chem., 265: branes. Mol. Membr. Biol., 12: 59–64, 1995. 1–4, 1990. 51. Teegarden, D., Taparowsky, E. J., and Kent, C. Altered phosphatidylcholine metab- 33. Walsh, J. P., Suen, R., and Glomset, J. A. Arachidonoyl-diacylglycerol kinase. olism in C3H10T1/2 cells transfected with the Harvey-ras oncogene. J. Biol. Chem., Specific in vitro inhibition by polyphosphoinositides suggests a mechanism for 265: 6042–6047, 1990. regulation of phosphatidylinositol biosynthesis. J. Biol. Chem., 270: 28647–28653, 52. Cuadrado, A., Carnero, A., Dolfi, F., Jimenez, B., and Lacal, J. C. Phosphorylcholine: 1995. a novel second messenger essential for mitogenic activity of growth factors. Onco- 34. Lykidis, A., Jackson, P. D., Rock, C. O., and Jackowski, S. The role of CDP- gene, 8: 2959–2968, 1993. diacylglycerol synthetase and phosphatidylinositol synthase activity levels in the 53. Jimenez, B., del Peso, L., Montaner, S., Esteve, P., and Lacal, J. C. Generation of regulation of cellular phosphatidylinositol content. J. Biol. Chem., 272: 33402– phosphorylcholine as an essential event in the activation of Raf-1 and MAP-kinases 33409, 1997. in growth factors-induced mitogenic stimulation. J. Cell Biochem., 57: 141–149, 35. Florin-Christensen, J., Florin-Christensen, M., Delfino, J. M., Stegmann, T., and 1995. Rasmussen, H. Metabolic fate of plasma membrane diacylglycerols in NIH 3T3 54. Hernandez-Alcoceba, R., Saniger, L., Campos, J., Nunez, M. C., Khaless, F., Gallo, fibroblasts. J. Biol. Chem., 267: 14783–14789, 1992. M. A., Espinosa, A., and Lacal, J. C. Choline kinase inhibitors as a novel approach 36. Kent, C. Eukaryotic phospholipid biosynthesis. Annu. Rev. Biochem., 64: 315–343, 1995. for antiproliferative drug design. Oncogene, 15: 2289–2301, 1997. 55. Daly, P. F., Lyon, R. C., Faustino, P. J., and Cohen, J. S. Phospholipid metabolism in 37. Kearns, B. G., Alb, J. G. J., and Bankaitis, V. Phosphatidylinositol transfer proteins: 31 the long and winding road to physiological function. Trends. Cell Biol., 8: 276–282, cancer cells monitored by P NMR spectroscopy. J. Biol. Chem., 262: 14875–14878, 1998. 1987. 31 38. Monaco, M. E., Alexander, R. J., Snoek, G. T., Moldover, N. H., Wirtz, K. W., and 56. Merchant, T. E., Gierke, L. W., Meneses, P., and Glonek, T. P magnetic resonance Walden, P. D. Evidence that mammalian phosphatidylinositol transfer protein regu- spectroscopic profiles of neoplastic human breast tissues. Cancer Res., 48: 5112– lates phosphatidylcholine metabolism. Biochem. J., 335: 175–179, 1998. 5118, 1988. 39. Jamal, Z., Martin, A., Gomez-Munoz, A., and Brindley, D. N. Plasma membrane 57. Ruiz-Cabello, J., and Cohen, J. S. Phospholipid metabolites as indicators of cancer fractions from rat liver contain a phosphatidate phosphohydrolase distinct from that cell function. NMR Biomed., 5: 226–233, 1992. in the endoplasmic reticulum and cytosol. J. Biol. Chem., 266: 2988–2996, 1991. 58. de Certaines, J. D., Larsen, V. A., Podo, F., Carpinelli, G., Briot, O., and Henriksen, 40. Marucci, L., Varticovski, L., and Arias, I. M. Effect of a xanthine analog on human O. In vivo 31P MRS of experimental tumours. NMR Biomed., 6: 345–365, 1993. hepatocellular carcinoma cells (Alexander) in culture and in xenografts in SCID mice. 59. Smith, T. A., Bush, C., Jameson, C., Titley, J. C., Leach, M. O., Wilman, D. E., and Hepatology, 26: 1195–1202, 1997. McCready, V. R. Phospholipid metabolites, prognosis and proliferation in human 41. Gennis, R. B. Biomembranes: Molecular Structure and Function, pp. 36–84. New breast carcinoma. NMR Biomed., 6: 318–323, 1993. York: Springer-Verlag, 1997. 60. Dalton, W. S. Mechanisms of drug resistance in hematologic malignancies. Semin. 42. Cullis, P. R., Hope, M. J., and Tilcock, C. P. Lipid polymorphism and the roles of Hematol., 34: 3–8, 1997. lipids in membranes. Chem. Phys. Lipids, 40: 127–144, 1986. 61. Kuwano, M., Toh, S., Uchiumi, T., Takano, H., Kohno, K., and Wada, M. Multidrug 43. DeKruijff, B. Polymorphic regulation of membrane lipid composition. Nature (Lond.), 329: 587–588, 1987. resistance-associated protein subfamily transporters and drug resistance. Anticancer 44. Bloom, M., Evans, E., and Mouritsen, O. G. Physical properties of the fluid lipid- Drug Des., 14: 123–131, 1999. bilayer component of cell membranes: a perspective. Q. Rev. Biophys., 24: 293–397, 62. Anthony, M. L., Zhao, M., and Brindle, K. M. Inhibition of phosphatidylcholine 1991. biosynthesis following induction of apoptosis in HL-60 cells. J. Biol. Chem., 274: 45. Liscovitch, M., and Chalifa, V. Signal activated phospholipase D. In: Mordechai 19686–19692, 1999. Liscovitch (ed.), Signal-activated Phospholipases, pp. 31–64. Austin: R. G. Landes 63. Kelland, L. Platinum complexes. In: B. A. Teicher (ed.), Cancer Therapeutics: Ex- Company, 1994. perimental and Clinical Agents, pp. 93–112. Totowa, NJ: Humana Press, 1997.

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Robert E. Finney, Edward Nudelman, Thayer White, et al.

Cancer Res 2000;60:5204-5213.

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