(CANCER RESEARCH 52, 2835-2840. May 15, 1992] Selective Inhibition of Phosphatidylinositol Phospholipase C by Cytotoxic Ether Lipid Analogues1

G. Powis,2 M. J. Seewald, C. Gratas, D. Melder, J. Riebow, and E. J. Modest

Department of Pharmacology. Mayo Clinic and Foundation, Rochester, Minnesota 55905 [G. P., M. J. S., C. G., D. M., J. R.J, and Department of Biochemistry, Boston University School of Medicine, Boston, Massachusetts 021 IS [E. J. M.]

ABSTRACT phate formation could be due to the ether lipid analogues The ether lipid analogue l-octadecyl-2-methyl-rac-glycero-3-phospho- depleting PIP2 as the substrate for inositol phosphate forma choline (ET-18-OCH.,) has been shown to be a direct inhibitor of Swiss tion, enhancing the breakdown of inositol phosphates, or inhib 3T3 fibroblast and BG1 ovarian adenocarcinoma cell cytosolic phosphoi- iting the PIPLC-mediated hydrolysis of PIP2. nositide selective phospholipase C (PIPLC) using |3H|-phosphatidylino- Hydrolysis of PIP2 by PIPLC is an important step mediating sitol-(4, 5)-bisphosphate (|'II|I"II'<) as the substrate. The inhibition the actions of growth factors and oncogenes on cell proliferation occurred when ET-18-OCH., was incorporated into the |3H|PIP2 substrate (7-10). A number of PIPLC isoforms have been described and micelles, with 50% inhibition «!(.,„)occurringat a I I 1K-(K II,: there are at least 4 genes, designated a, ß,7, and 5 (11, 12). |3H|PIP2 ratio of 0.04, or an assay concentration of 0.4 MM,and when Differential expression of some PIPLC isoforms between nor ET-18-OCH., was added directly to the incubation, with an ICs>of 9.6 mal and tumor cells has been reported (13-15). Other phos- MM.Lipid prepared from cells exposed to cytotoxic concentrations of ET- 18-OCH, for 18 h also inhibited PIPLC with an 1C»< 1 MM.The pholipases such as PCPLC and PLD have also been implicated as playing a role in cell proliferation (16-18). We now report noncytotoxic analogue 1-O-alkyl-l-hydroxy-sn-glycero^-phosphocho- line inhibited PIPLC when incorporated into the |3H|PIP2 substrate that cytotoxic ether lipid analogues are potent inhibitors of micelles, but lipid from cells grown with 5 MMl-O-alkyl-2-hydroxy-í/i- PIPLC but weak inhibitors of PLD and PCPLC. The inhibition glycero-3-phosphocholine did not inhibit PIPLC. BG1 cells, which were of PIPLC by the ether lipid analogues, together with other more sensitive than Swiss 3T3 fibroblasts to growth inhibition by ET- effects on intracellular signaling, could contribute to the 18-OCH.,, had a cytosolic PIPLC activity one-third that of Swiss 3T3 growth-inhibitory activity of this class of compounds. cells. NIH 3T3 cells exhibited the same sensitivity to growth inhibition by ET-18-OCHj as Swiss 3T3 cells and had a similar level of PIPLC. v- .v/.vNIH 3T3 cells were relatively resistant (>3-fold) to growth inhibition MATERIALS AND METHODS by ET-18-OCHj and had a cytosolic PIPLC activity more than twice that of the wild type cells. ET-18-OCH., was a weak inhibitor, K .„>100 Murine Swiss 3T3 fibroblasts and NIH 3T3 cells were purchased from the American Tissue Type Culture (Rockville, MD). v-í/í-trans- MM,of phospholipase D activity in NIH 3T3 cell membranes. In intact NIH 3T3 cells ET-18-OCH., at cytotoxic concentrations did not inhibit formed NIH 3T3 cells were obtained from Dr. S. Aaronson (National phospholipase D or phosphatidylcholine-selective phospholipase C activ Cancer Institute, Bethesda, MD). BG1 ovarian adenocarcinoma cells (19) were provided by Dr. E. Modest. Cells were maintained in Corning ity. The results show that the ether lipid analogues at cytotoxic concen 75-cm2 culture flasks (Corning, NY) in DMEM containing 10% fetal trations are selective inhibitors of PIPLC and that the inhibition of calf serum and harvested for passage with 0.05% trypsin and 0.5 mM PIPLC may be related to the growth inhibitory activity of the ether lipid EDTA. The ether lipid analogues used were ET-18-OCH., provided by analogues. Dr. E. Modest, CP10 (20) provided by Dr. Claude Piantadosi (Univer sity of North Carolina, Chapel Hill, NC), PAF and l-O-alkyl-2-hy- INTRODUCTION droxy-în-glycero-3-phosphocholine obtained from Avanti Polar Lipids (Alabaster, AL). U-73122 was provided by Dr. G. Bundy, The Upjohn The ether lipid analogues of PAF3-related compounds are Company (Kalamazoo, MI), and D-609 by Dr. G. Quack, Merz and members of a new class of DNA-noninteractive antitumor Co. (Frankfurt, Germany). Choline chloride, phosphocholine chloride, agents (1, 2). They cause induction of cellular differentiation cytidine 5'-diphosphocholine, and Dowex 50-X8-400 H* were ob (3), activate cytotoxic macrophages (4), and exhibit direct anti- tained from Sigma Chemical Co. (St. Louis, MO). [3H]-methylcholine proliferative activity against tumor cells (1,5). chloride, 85 mCi/mmol, and [2-3H]-D-myo-inositol(l,4)bisphosphate ([3H]PIP2, 1 Ci/mmol, were obtained from American Radiochemicals, We have recently shown that antitumor ether lipid analogues inhibit growth factor-dependent intracellular free Ca2+signaling Inc. (St. Louis, MO). (6). They block the inositol(l,4,5)trisphosphate-induced release Cell growth inhibition was measured as described previously with of Ca2+ from intracellular stores as well as inhibiting the hy the cells growing on plastic surfaces (6). PIPLC was assayed by a modification of the method of Wahl et al. (21). Briefly, cells drolysis of PIP2 to inositol phosphates stimulated by PDGF were homogenized at 4°Cin medium containing 10 mM 4-(2-hydroxy- and by fluoroaluminate anión.This inhibition of inositol phos- ethyl)-l-piperazineethanesulfonic acid (pH 7.0), 5 mM [ethylene- bis(oxyethylenenitrilo)]tetraacetic acid, 1 mM MgCl2, 0.2 mM dithio- Received 11/12/91; accepted 3/9/92. The costs of publication of this article were defrayed in part by the payment threitol, and 0.5 mM PMSF, centrifugea at 600 x g for 10 min and of page charges. This article must therefore be hereby marked advertisement in then at 100,000 x g for 40 min, and the supernatant was diluted to 200 accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Mgprotein/ml. ['HJPIPj substrate micelles were prepared from a chlo 1Supported by NIH Grants 42286 and 48725 (G. P.) and CA41314 (E. J. M.). 2To whom requests for reprints should be addressed, at Arizona Cancer Center, roform solution containing 48 nmol (0.5 MCi)[3H]PIP2, with or without University of Arizona, 1501 North Campbell Ave., Tucson, AZ 85724. varying amounts of the ether lipid analogues or other lipids, which was 3The abbreviations used are: PAF, platelet-activating factor (I-alkyl-2-acetyl- dried under N2 and then sonicated with 1 ml 50 mM methyleneetha- sn-glycero-3-phosphocholine); PIPLC, phosphatidylinositol-selective phospholi pase C; PCPLC, phosphatidylcholine-selective phospholipase C; PLD, phospho nesulfonate buffer (pH 7.0), 2 mM [ethylenebis(oxyethylenenitrilo)] lipase D; P1P2, phosphatidylinositol(4,5)bisphosphate; PDGF, platelet-derived tetraacetic acid, and 0.1% octyl-/3-D-glucopyranoside, followed by the growth factor; ET-18-OCHj, l-octadecyl-2-methyl-rac-glycero-3-phosphocho- addition of 2 ml of the same buffer containing 1.12 mM CaCl2. Sixty line; CP10, l-5-octadecyl-2-O-methyl-i-thiopropyl-3-A',JV-dimethylhydroxypro- n\ of the [3H]PIP2 substrate solution were incubated with 40 M' of pyl ammonium iodide; U-73122, l-||6-|[I7/j-3-methoxyestra-l,3,5(10)-trien-17- cytosolic fraction containing 5 MBof protein for 10 min at 37°C.In yl]amino|hexyl||-lA/-pyrrole-2,5-dione; D-609, tricyclodecan-9-ylxanthogenate; DMEM, Dulbecco's modified Eagle's medium; PMSF, phenylmethylsulfonyl some studies the ether analogues were added directly to the incubation fluoride; IC50,50% inhibitory concentration. mixture. The incubation was terminated with 0.1 ml 10% bovine serum 2835

Downloaded from cancerres.aacrjournals.org on September 23, 2021. © 1992 American Association for Cancer Research. ETHER LIPID INHIBITION OF PHOSPHOLIPASE C albumin and 0.5 ml 10% trichloroacetic acid. After standing for l h at 4°C,the mixture was centrifugea at 18,000 x g for 5 min and 0.4 ml of the supernatant was taken for liquid scintillation counting. Prelimi nary studies showed that [3H]PIP2 hydrolysis was linear with protein up to 8 //ii sample and linear with time up to 15 min. PCPLC and PLD activity in intact NIH 3T3 were measured by growing 2 x IO5cells in 35-mm culture dishes with ['H]methylcholine chloride, 1.5 iiCi/ml, for 48 h. The cells were washed twice for 30 min with DMEM at 37°Cand then incubated with DMEM with 0.1% bovine serum albumin, with or without ether lipid, for 30 min. The incubation medium was replaced with 1 ml ice cold methanol, and the cells were liberated by scraping. The mixture was extracted with 4 ml chloroform:methanol (1:1, v:v) and then back extracted with an addi tional 3 ml water (22). Radioactivity in the aqueous phase of the cell extract and in the incubation medium was separated into choline, phosphocholine, and glycerophosphocholine by chromatography on Dowex 50-X8-400 H* as described by Cook and Wakelam (23). To 0.00 0.02 0.04 0.06 0.08 0.10 measure membrane phospholipase activity NIH 3T3 cells were grown with ['H]methylcholine chloride for 48 h, washed and homogenized in Ratio ( -1] 20 HIM Tris-buffer (pH 7.5), 2 mM MgCl2 containing the protease inhibitors 0.1 mivi leupeptin and 1 miviPMSF. A microsomal fraction Fig. 1. Inhibition of PIPLC activity by ET-18-OCH,. PIPLC activity in the cytosol of Swiss 3T3 fibroblasts (O) and BG1 ovarian adenocarcinoma cells was prepared by the method of Kiss and Anderson (24). Incubations (•)was measured with |3H)-P1P2 as substrate. ET-18-OCHj was mixed with were conducted with 50 Mgmicrosomal protein in 0.1 ml 40 mivi Tris (3H]PIP2 to form the substrate micelles at the ratio shown. Each point is the buffer (pH 7.0), 5 mM MgCl2, 0.05 mM leupeptin, 0.5 mM PMSF, 1 mean of six determinations; bars, SD. mM choline, 1 mM phosphocholine, and 0.2 mM cytidine 5'-diphos- phocholine at 37°for 1 h. The reaction was stopped with 0.1 ml 10% Table 2 Inhibition of PIPLC by ether lipidi bovine serum albumin and 0.5 ml 25% trichloroacetic acid. After PIPLC in cytosol prepared from Swiss 3T3 fibroblasts was measured with centrifugation at 7000 x g for 5 min at 4°C,an aliquot of the super detergent solubilized |'H]PIP2 micelles as the substrate. Ether lipids were incor natant was taken for the determination of total radioactivity. Phospha- porated with ['H]PIP2 into the micelles at a ratio of 1:10. Enzyme activity was tidylcholine metabolites in the remaining supernatant were measured measured as described in the text. Values are mean ±SD of six determinations. Numbers in parentheses, percentage of the control value. as described previously. PIPLC Lipid (nmol/min/mg) RESULTS Control ±0.04 (100) ET-18-OCH, 0.27 ±0.05° (13) Cell Growth Inhibition by ET-18-OCH,. BG1 ovarian ade- CP10 0.05 ±0.07° (3) nocarcinoma cells were more sensitive to growth inhibition by LysoPAF* 0.10 + 0.11° (5) the ether lipid analogue ET-18-OCHj than Swiss 3T3 fibro- PAF 1.70 ±0.27 (87) Phosphatidylcholine1.96 1.77 ±0.25 (90) blasts, either at 1 h or with continuous drug exposure (Table ' P < 0.05 compared to the control value. 1). The sensitivity of Swiss 3T3 cells and BG1 cells to ET-18- ' PAF, l-alkyl-i-acetyl-Jn-glycerophosphocholine. OCHj with continuous exposure was similar to that previously reported (6, 19, 25). Both cell lines showed greater sensitivity to 1 h exposure to ET-18-OCHj in the absence than in the hydrolysis (IC50) ±SD was 0.040 ±0.005 with Swiss 3T3 cell PIPLC and 0.045 ±0.012 with BG1 cell PIPLC. These values presence of serum in the medium. This is probably due to the represent assay IC50s for ET-18-OCH., of 0.4 ±0.1 MMwith extensive plasma protein binding of ET-18-OCH3 that reduces Swiss 3T3 cell PIPLC and 0.4 ±0.1 with BG1 cell PIPLC. the free concentration of the drug in the medium (26). Adding ET-18-OCHj directly to the incubation was less effec Inhibition of PIPLC by Ether Lipids. ET-18-OCH, incorpo rated into the [3H]PIP2 substrate micelles was an inhibitor of tive at inhibiting Swiss 3T3 cell PIPLC with an IC50 of 9.6 ± [1H]PIP2 hydrolysis by cytosolic PIPLC from both Swiss 3T3 1.2 MM.This is probably because ET-18-OCH3 is only slowly incorporated into the [3H]PIP2 micelles when added directly to fibroblasts and BG1 ovarian adenocarcinoma cells (Fig. 1). The ratio of ET-18-OCHj to [3H]PIP2 causing 50% inhibition of the incubation. The cytotoxic thioalkyl ether lipid CP10 as well as the noncytotoxic PAF were inhibitors of Swiss 3T3 cell PIPLC (Table 2). Phosphatidylcholine and PAF did not inhibit Table 1 Inhibition of cell growth by ET-18-OCH, [3H]PIP2 hydrolysis showing that incorporation of a phospho- Cells plated 24 h previously were exposed to ET-18-OCH., at various concen lipid into the [3H]PIP2 micelles alone was not sufficient to trations in DMEM, with or without 10% fetal calf serum (PCS), for 1 h, washed, and grown for 7 days in DMEM with 10% PCS or exposed continuously to ET- inhibit PIPLC activity. ET-18-OCH3 was a potent inhibitor of 18-OCH, in DMEM with 10% PCS for 7 days. The cells were then counted and Swiss 3T3 cell membrane-bound PIPLC with IC50 < 0.1 MM the concentration of ET-18-OCH, required to produce 50% inhibition of cell when incorporated into the [3H]PIP2 substrate micelles and an growth (ICso) ±SD was compared to that of cells not treated with ET-18-OCH,, calculated by nonlinear least squares regression of the data. The results are from IC50 of 5 MMwhen added directly to the assay (results not a typical experiment. shown). hIC50(>»M)1.0 IC50 Comparison with Other PIPLC Inhibitors. The activity of the (MM)ND° lineSwiss Cell ether lipid analogues as inhibitors of PIPLC was compared 3T3 fibroblasts with other PIPLC inhibitors (Table 3). U-73122 is an inhibitor -PCS + 0.2 +FCSBG1 62.5 ±12.11.5 19.0 ±1.3ND0.4 of phospholipase C-dependent processes in human platelets and polymorphonuclear neutrophils (27, 28). D-609 has been ovarian adenocarcinoma reported to inhibit phospholipase C-dependent activities in -PCS ±0.4 +FCS1±0.1" 10.2 ±5.8Continuous hamster fibroblasts (29). It is interesting that, under acid con ND, not determined. ditions, D-609 can reverse the transformed phenotype of some 2836

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Table 3 Comparison ofET-18-OCH, with other inhibitors of phospholipase C whereas cells with low levels of the enzyme are more sensitive. The inhibition of PIPLC in Swiss 3T3 cell cytosol using |'H]PIP2 as a substrate Inhibition of Other Phospholipases. Cell membranes prepared was measured. Inhibitors were added directly to the incubation mixture or incorporated into the [3H|PIP2 micelles. Enzyme activity was measured as de from [3H]choline-labeled NIH 3T3 cells released radiolabeled scribed in the text. Values are expressed as the 1C»(±SD) of the drug concen choline, phosphocholine, and small amounts of glycerophos- tration in the assay obtained by nonlinear least square regression analysis of the phocholine upon incubation at 37°C(Fig. 2). NIH 3T3 cell concentration response data. membranes have been reported previously to contain PLD (24, directly ICM IC50(MM)0.4 CompoundET-18-OCH,"U-73122(MM)9.6 33), which gives choline as the product of phosphatidylcholine ±1.2 ±0.1 hydrolysis. The formation of phosphocholine seen in the pres 11.2±O.IND*" 101 ±8 ent study suggests there is also PCPLC in NIH 3T3 cell D-609Added 1046 ±27Incorporated membranes. ET-18-OCH, was a weak inhibitor of membrane Data from Fig. 1. * ND, not determined. phosphatidylcholine hydrolysis with an IC50 >100 MM (the highest concentration tested). Intact NIH 3T3 cells labeled with [3H]choline were found to Table 4 Cytosolic PIPLC in Swiss 3T3 and BG1 cells exposed to ET-18-OCH, contain appreciable amounts of radiolabeled phosphocholine Swiss 3T3 fibroblasts or BGI ovarian adenocarcinoma cells were grown for 18 h in the presence of ether lipid. Cytosolic PIPLC activity was measured with [3H] and small amounts of choline at t —¿Omin (Fig. 3). Extensive PIPj as the substrate, as described in the text. Values are mean ±SD of six washing of the cells before the study did not lower the levels of determinations. phosphocholine or choline which were, trapped with the cells. In the absence of agents to stimulate phosphatidylcholine hy CellsSwiss drolysis, ET-18-OCH, at concentrations above 20 MMproduced 3T3 fibroblast cytosol Control ±0.31 a small increase in the formation of both choline and phospho ET-18-OCH,BGIGrown with 10 MM 4.26 +0.511.42 choline. At the same time, there was a redistribution of water- soluble radioactivity from inside the cell to the extracellular ovarian adenocarcinoma cytosol ±0.35° Control medium, presumably reflecting an increase in cell membrane Grown±0.24°° with 5 MMET-18-OCH,PIPLC(nmol/min/mg)3.88 1.45 permeability at high concentrations of ET-I8-OCH,. P < 0.05 compared to value for Swiss 3T3 cells.

DISCUSSION tumor cells (30). ET-18-OCH, proved to be a considerably more potent inhibitor of PIPLC, whether added directly to the Our previous studies have shown that the ether lipid analogue assay or incorporated into the [3H]PIP2 substrate micelles, than ET-18-OCH, inhibits the formation of inositol phosphates by either U-73122 or D-609. Neomycin sulfate, which binds with Swiss 3T3 fibroblasts stimulated with either PDGF or fluo- high affinity to PIP2 (31), completely inhibited PIPLC activity roaluminate anión(6). PDGF increases inositol phosphate for at 1 HIM(results not shown). mation by the PDGF receptor-mediated tyrosine phosphoryla- PIPLC Activity in ET-18-OCH,-exposed Cells. There was no tion of a specific PIPLC, PIPLC-7, (34, 35). Fluoroaluminate effect on cytosolic PIPLC activity when Swiss 3T3 fibroblasts anión stimulates inositol phosphate formation through the G or BG1 ovarian adenocarcinoma cells were grown with a cyto- protein-mediated activation (36) of another PIPLC, PIPLC-/3i toxic concentration of ET-18-OCH, for 18 h (Table 4). (37). The present work shows that ET-18-OCH, and other PIPLC Inhibition by Lipids from Ether Lipid-exposed Cells. ether lipid analogues are potent inhibitors of both cystosolic Ether lipid analogues are known to be incorporated in cell and membrane bound PIPLC. PIPLC-7i is the predominant membranes (32). In order to determine whether the ether lipid PIPLC isoform in the cytosol of fibroblasts (11), while PIPLC- analogues present in the cellular lipid from cells exposed to /3, is over 80% membrane bound (38, 39). ET-18-OCH, was a cytotoxic concentrations of the analogues could inhibit PIPLC more potent inhibitor of membrane-associated PIPLC activity activity, cells were grown for 18 h with the analogues and the than cytosolic PIPLC, suggesting there may be some isoform inhibitory effect of the extracted cell lipids on [3H]PIP2 hydrol specificity to the inhibition. This possibility, however, requires ysis by PIPLC studied (Table 5). Lipid from both Swiss 3T3 further investigation. fibroblasts and BG 1 ovarian adenocarcinoma cells grown with 1 to 10 MMET-18-OCH, for 18 h inhibited PIPLC activity. Table 5 Effect of cellular lipids on PIPLC Inhibition of PIPLC activity was also seen with lipid from Swiss Swiss 3T3 fibroblasts or BG 1 ovarian adenocarcinoma cells were grown for 18 3T3 cells grown with 5 MMCP10, but not with lipid from cells h in the absence or presence of ether lipids and total cell lipids extracted. One grown with 5 MMPAF. mg of this lipid was mixed with 48 nmol |'H]PIP2 used to prepare the substrate micelles. PIPLC activity in the cytosol of nontreated Swiss 3T3 cells was measured PIPLC Activity and Growth Inhibition. BGI ovarian adeno using these micelles. Enzyme activity was measured as described in the text. carcinoma cells, which were considerably more sensitive than Values are mean ±SDof six determinations. Numbers in parentheses, percentage Swiss 3T3 cells to growth inhibition by ET-18-OCH., (Table of the appropriate control value. 1), had a cytosolic PIPLC activity 37% that found in Swiss 3T3 PIPLC cells (Table 4). In order to further investigate the relationship Treatment (nmol/min/mg) between cell growth inhibition and PIPLC activity, NIH 3T3 Swiss 3T3 cell lipid Nontreated 2.64 ±0.14 (100) cells and v-sw-transformed NIH 3T3 cells were studied. The ET-18-OCH,, 1 MMfor 18 h 0.51 ±0.08°(19) ICsoS for growth inhibition by ET-18-OCH, (with continuous ET-18-OCH.,, 5 MMfor 18 h 0.36 ±0.12°(14) ET-18-OCH,, 10 MMfor 18 h 0° (0) exposure) were 20.1 ±4.1 MMfor NIH 3T3 cells and 74.3 ± CP10, 5 MMfor 18 h 0.19 ±0.08° (7) 4.1 MMfor \-sis NIH 3T3 cells. Cytosolic PIPLC activity was PAF, 5 MMfor 18 h 2.59 ±0.09 (98) 4.1 ±0.03 nmol/min/mg in NIH 3T3 cells and 10.16 ±0.39 BGI cell lipid Nontreated 2.61 ±0.19 (100) nmol/min/mg in v-sis NIH 3T3 cells (P < 0.01). Thus, it ET-18-OCH,, 1 MMfor 18 h 0.27 ±0.08°(10) appears that cells with high levels of cytosolic PIPLC are 1P < 0.05 compared to the appropriate control. relatively resistant to growth inhibition by ET-18-OCH,, *PAF, l-alkyl-i-acetyl-sn-glycero-S-phosphocholine. 2837

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25 50 75 100 To Control ET-1B-OCH- ET-18-OCH3 (nil) 100/41 Fig. 2. Effect of ET-18-OCH3 on the release of radioactivity from [3H]choline-labeled NIH 3T3 cell membranes. Cells were grown for 48 h with [3H]choline and a microsomal fraction was prepared. Left, the release of total radioactivity was measured over l h incubation at 37'C with different concentrations of ET-18-OCH3. Values are the mean of 3 determinations; han. SD. Right, separation of the radioactivity into choline (ED,phosphocholine (LJ).and glycerophosphocholine (D); the results shown are a typical experiment. 7"0,water-soluble radioactivity before incubation; control, l h at 37*C; ET-18-OCH3, with 100 /IM ET-18-OCH3 for l h at 37-C.

Fig. 3. Effect of ET-18-OCHj on the for mation of water-soluble radioactivity by I'll] choline-labeled NIH 3T3 cells. The cells were labeled with ['HJcholine for 48 h and washed, and the formation and redistribution of water- soluble radioactivity was measured over 30 min at 37'C. Left, formation of choline (ED and phosphocholine (D), showing the results of a typical experiment. l\,. zero time. The other values are after 30 min with 0, 20, and 80 /j\i ET-18-OCH3. Right, total water-soluble radio activity present after 30 min incubation (V); water-soluble radioactivity associated with the cells (•);water-soluble radioactivity in the in cubation medium (O). Values are mean of 3 determinations; bars, SD.

O '¿OpU 20 40 60 ET-18-OCH, ET-18-OCHS (luí)

The assay for PIPLC uses two phases, aqueous and lipid, and amount of ether lipid accumulated by the cell membrane should it is difficult to know the actual concentration of ether lipid be sufficient to inhibit PIP2 hydrolysis by PIPLC. However, a analogues causing inhibition of [3H]PIP2 hydrolysis. Although direct demonstration that growth-inhibitory concentrations of PIPLC-7 is a soluble enzyme PIPLC must bind to the cell ether lipid analogues can inhibit PIPLC was provided by our membrane or to the substrate micelles before hydrolyzing PIP2 finding that lipid extracted from cells grown with ether lipid (40). When ET-18-OCH3 was incorporated into the [3H]PIP2 analogues inhibited PIPLC. It appears that the ether lipid micelles its IC50 was at a nominal assay concentration of 0.4 analogues, or their metabolites, are incorporated into the cell /iM. When ET-18-OCH3 was added directly to the incubation membrane at sufficient concentrations to inhibit PIPLC. The mixture its IC50 9.6 UM. The difference in potency may be ether lipid analogues are lipid phase inhibitors of PIPLC and because a smaller amount of ET-18-OCH3 becomes incorpo do not inhibit PIPLC in the aqueous phase, and in cells exposed rated into the [3H]PIP2 micelles when the ether lipid is added to ET-18-OCH3 for 18 h there was no change in cytosolic directly to the incubation mixture. A preliminary report (41) PIPLC. has described inhibition of human melanoma-derived PIPLC The mechanism for inhibition of PIPLC by the ether lipid by phosphatidylinositol ether lipid analogues, but only at con analogues is unknown. ET-18-OCH3 is only slowly metabolized centrations above the IC5o for inhibition of cell growth. An by cells (44, 45) so that the inhibition of PIPLC is likely to be important question for us to answer, therefore, was whether a direct effect of ET-18-OCH3 rather than by a metabolite. PIPLC could be inhibited at concentrations of ether lipid ana PAF, on the other hand, is rapidly metabolized (45, 46) which logues likely to be achieved in the lipid phase of cells exposed could explain the lack of PIPLC inhibitory activity of lipid to growth-inhibitory concentrations of the analogues. Accu from PAF-exposed cells, despite the direct inhibitory effects of mulation of ET-18-OCH3 has been reported to approach 17% PAF on the enzyme. of the total phospholipids of purified cell plasma membranes Phospholipases in addition to PIPLC, including PCPLC and (32) whereas PIP2 represents only a small percentage of mem PLD, may play an important role in cell proliferation (16, 47, brane phospholipids (42, 43). It appears, therefore, that the 48). The specificity of the inhibition of PIPLC by the ether 2838

Downloaded from cancerres.aacrjournals.org on September 23, 2021. © 1992 American Association for Cancer Research. ETHER LIPID INHIBITION OF PHOSPHOLIPASE C lipid analogues compared to PCPLC and PLD was studied. reticulum Ca2+ uptake, and inhibition of inositol phosphate- ET-18-OCHj was found to be a weak inhibitor of PCPLC and mediated Ca2+ release from endoplasmic reticulum stores (6). PLD in NIH 3T3 cell membranes. At concentrations of ET- We have previously proposed that the action of the ether lipid 18-OCH3 above cytotoxic levels there was even a small stimu analogues on intracellular signaling, perhaps at several sites, lation of PLD and PCPLC activity in the intact cells. Although could be responsible for their cell growth-inhibitory activity (6). we have tentatively identified the enzymes responsible for phos- The present work shows that PIPLC could be one of the sites phatidylcholine hydrolysis as PIPLC and PLD, because of the inhibited by ether lipid analogues contributing to their ability possibility of product interconversion the identity of the en to inhibit cell growth. zymes involved in phosphatidylcholine hydrolysis cannot be conclusively established by product identification alone (16). REFERENCES High concentrations of ET-18-OCH3 caused a redistribution of choline and phosphocholine from inside the cell to the extra 1. Berdel, W. E., and Munder, P. G. Antineoplastic actions of ether lipids related to platelet-activating factor. In: F. Snyder (ed.), Platelet-activating cellular medium. This redistribution probably reflects an in Factor and Related Lipid Mediators, pp. 449-468. New York: Plenum crease in cell membrane permeability caused by ET-18-OCH3. Publishing Corp., 1987. Ether lipids are known to result in the fluidization of lipid 2. Modest, E. J., Noseda, A., Daniel, L. W., and Piantadosi, C. Membrane- membranes (49, 50), and we have previously reported that ET- targeted ether lipid analogs with antineoplastic activity. Cancer Chemother. Pharmacol., 24 (Sappi 2): 359, 1989. 18-OCH3 at concentrations above cytotoxic concentrations 3. Ilimili«.Y., Kasukabe, T., Hozumi, M., Tsushima, S., and Nomura, H. causes an increase in cell membrane permeability measured by Cytotoxicity and metabolism of alkyl phospholipid analogues in neoplastic the influx of Ca2+(6). cells. Cancer Res., 46: 5803-5809, 1980. 4. Berdel, W. E., Bausert, W. R., Weltzien, H. U., Modolell, M. L., Widmann, Differences in the sensitivity of cell lines to growth inhibition K. H., and Munder, P. The influence of alkyl-phospholipids and phospho- lipid-activated macrophages on the development of metastasis of 3-Lewis- by ether lipid analogues have been variously ascribed to differ lung carcinoma. Eur. J. Cancer, 16:1199-1204, 1980. ences in the cellular incorporation of ether lipids (44, 51), 5. Andressen, R., Modolell, M., Weltzien, H. U., Eibl, H., Common, H. H., perhaps involving an endocytic process (52), to the cellular Löhr,G. W., and Munder, P. G. Selective destruction of human leukemic content of endogenous ether-linked lipids (53) and cholesterol cells by alkyl-phospholipids. Cancer Res., 38: 3894-3899, 1978. 6. Seewald, M. J., Olsen, R. A., Sehgal, I., Melder, D. C., Modest, E. J., and (54), and to metabolism of the ether lipid analogues (32). None Powis, G. Inhibition of growth factor-dependent inositol phosphate ( V " of these mechanisms alone appears sufficient to account for signalling by antitumor ether lipid analogues. Cancer Res., 50: 4458-4463, 1990. differences in the sensitivity of cells to the ether lipid analogues. 7. Tucker, R. W., Chang, D. T., and Meade-Cobun, K. Effects of platelet- BG1 human ovarian adenocarcinoma cells showed an increased derived growth factor and fibroblast growth factor on free intracellular sensitivity to growth inhibition by ET-18-OCH3 compared to calcium and mitogenesis. J. Cell. Biochem., 61: 203-212, 1989. 8. Cantley, L. C., Auger, K. R., Carpenter, C., Duckworth, B., Graziani, A., Swiss 3T3 fibroblasts. The PIPLC activity from Swiss 3T3 cells Kapeller, R., and Sditoli'. S. Oncogenes and signal transduction. Cell, 64: and BG1 cells was equally sensitive to inhibition by ET-18- 281-302, 1991. OCH3. However, BG1 cells had only about one-half the cyto- 9. Martin, T. F. J. Receptor regulation of phosphoinositidase C. Pharmacol. Ther., 49: 329-345, 1991. solic PIPLC activity compared to Swiss 3T3 cells. NIH 3T3 10. Heikkila, R., Trepel, J. B., Cultitta, F., Neckers, L. M., and Sausville, E. A. cells had the same sensitivity to growth inhibition by ET-18- Beinihesin -related peptides induce calcium mobilization in a subset of human small cell lung cancer cell lines. J. Biol. (hem., 262: 16456-16460, 1987. OCH.i compared to Swiss 3T3 cells and had similar levels of cytosolic PIPLC. v-ji'j-transformed NIH 3T3 cells were rela 11. Kriz, R., Lin, L-L., Sultzman, L., Ellis, C., Heldin, C-H., Pawson, T., and Knopf, J. Phospholipase C isozymes: structural and functional similarities. tively resistant to growth inhibition by ET-18-OCH3 and had In: 1990 Proto-Oncogenes in Cell Development, Ciba Foundation Sympo sium, pp. 112-127. Chichester, United Kingdom: John Wiley and Sons, Inc., cytosolic PIPLC more than double that of NIH 3T3 or Swiss 1990. 3T3 cells. Whether such differences in PIPLC are sufficient to 12. Rhee, S. G., Suh, P-G., Ryu, S-H., and Lee, S. Y. Studies of inositol account for the difference in growth inhibition caused by the phospholipid-specific phospholipase C. Science (Washington DC), 244:546- 550, 1989. ether lipid analogues in these cell lines will require further 13. Haas, N., McDanel, H., Godwin, A., Humphrey, P., Bigner, S., and Wong, A. Specific expression of phospholipase ( ' -, in human glial tumors. Proc. investigation. PIPLC-mediated phosphoinositide metabolism is an impor Am. Assoc. Cancer Res., 32: 17, 1991. 14. Arteaga, C. L., Johnson, M., Coffey, R. J., and Todderud, G. Tyrosine tant step for growth factor stimulated cell proliferation. Mi- phosphorylated phospholipase C (I'M ) gamma 1 is coexpressed with EGF croinjection of PIPLC-/3 or -7 into quiescent NIH 3T3 cells receptors (R) or erbB-2 in malignant but not benign human breast epithelial cells. Proc. Am. Assoc. Cancer Res., 32:46, 1991. results in an acute dose-dependent induction of DNA synthesis 15. Rhee, S. G., Kim, H., Suh, P-G., and Choi, W. C. Multiple forms of and phenotypic transformation (55). Microinjection of antibod phosphoinositide-specific phospholipase C and different modes of activation. ies against PIPLC-7, but not PIPLC-/3, into NIH 3T3 cells Biochem. Soc. Trans., 19: 337-341, 1991. 16. Dennis, E. A., Rhee, S. G., Billah, M. M., and Hannun, Y. A. Role of blocks serum-stimulated and ros-stimulated DNA synthesis phospholipases in generating lipid second messengers in signal transduction. (28). Mutant Chinese hamster ovary cells lacking PIPLC-a and FASEB J., 5: 2068-2077, 1991. with an abnormal PIPLC-7 activity do not show increased 17. Wakelam, M. J. O., Cook, S. J., Currie, S., Palmer, S., and Plevin, R. Receptor regulation of intracellular phospholipases. Biochem. Soc. Trans., phosphatidylinositol turnover and cell proliferation in response 79:321-324, 1991. to thrombin as do wild-type Chinese hamster ovary cells (56). 18. Insel, P. A., Weiss, B. A., Slivka, S. R., Howard, M. J., Waite, J. J., and Godson, C. A. Regulation of phospholipase A2 by receptors in MDCK-DI Overexpression of PIPLC-7 in cells, although not transforming, cells. Biochem. Soc. Trans., 19: 329-333, 1991. results in an exaggerated breakdown of PIP? in response to 19. Noseda, A., Kerens. M. E., Piantodosi, C., and Modest, E. J. Neoplastic cell inhibition with new ether lipid analogs. Lipids, 22: 878-883, 1987. growth factors such as PDGF, but the cells do not show an 20. Marasco, C. J., Jr., Piantadosi, C., Ishaq, K. S., Meyer, K. L., Morris- enhanced mitogenic response (35, 57, 58). Thus, although Natschke, S., Small, G. W., and Daniel, L. W. Synthesis and biological PIPLC activity is not limiting for cell proliferation under nor activity of novel quaternary ammonium derivatives of alkylglycerols as potent inhibitors of protein kinase C. J. Med. Chem., 33:985-991, 1990. mal conditions, inhibition of PIPLC by agents such as the ether 21. Wahl, M. I., Daniel, T. O., and Carpenter, G. Antiphosphotyrosine recovery lipid analogues could lead to cell growth inhibition. However, of phospholipase C activity after EGF treatment of A-431 cells. Science the ether lipid analogues have effects on growth factor-signaling (Washington DC), 241:968-970, 1988. 22. Bligh, E., and Dyer, W. A rapid method for total lipid extraction and pathways in addition to the inhibition of PIPLC. These include purification. Can. J. Biochem. Physiol., 37: 911-917, 1959. inhibition of protein kinase C (59), inhibition of endoplasmic 23. Cook, S. J., and Wakelam, M. J. O. Analyses of the water soluble products 2839

Downloaded from cancerres.aacrjournals.org on September 23, 2021. © 1992 American Association for Cancer Research. ETHER LIPID INHIBITION OF PHOSPHOLIPASE C

of phosphatidylcholine breakdown by ¡on-exchange chromatography. 43 Michel!, R. H.. Kirk, C. J.. Jones, L. M., Dawnes, C. P., and Creba, J. A. Biochem. J., 263: 581-587, 1989. The stimulation of inositol metabolism that accompanies calcium mobiliza 24. Kiss, Z., and Anderson, W. B. Phorbol ester stimulates the hydrolysis of tion in stimulated cells: defined characteristics and unanswered questions. phosphatidyl ethanolamine in leukemic HL-60, NIH 3T3 and baby hamster Philos. Trans. R. Soc. Lond. B. 296: 123-137, 1981. kidney cells. J. Biol. Chem., 264: 1483-1487, 1989. 44. Hoffman, D. R., Hoffman, L. H., and Snyder, F. Cytotoxicity and metabolism 25. Wallen, C. A., Fujiwara, K.. and Modest, E. J. Cell kill and cytostasis by an of alkyl phospholipid analogues in neoplastic cells. Cancer Res., 46: 5803- ether lipid. Proc. Am. Assoc. Cancer Res., 31:409, 1990. 5809, 1986. 26. Powis, G., and Kozikowski, A. Growth factor and oncogene signalling 45 Wilcox, R. W., Wykle, R. L., Schmitt, J. D., and Daniel, L. W. The pathways as targets of rational anticancer drug development. Clin. Biochem., degradation of platelet-activating factor and related lipids: susceptibility to 2*385-397, 1991. phospholipases C and D. Lipids, 22: 800-807. 1987. 27. Bleasdale, J. E., Bundy, G. L., Bunting, S., Fitzpatrick, F. A., Huff, R. M., 46 Wykle, R. L., and Schremmer, J. M. Alkylphospholipase D pathway in the Sun, F. F., and Pike, J. E. Inhibition of phospholipase C-dependent processes metabolism of ether-linked lipids in brain microsomes. J. Biol. Chem., 249: by U-73,122. In: B. Samuelsson, P. Y-K. Wong, and F. F. Sun (eds.). 1742-1746, 1974. Advances in Prostaglandin, Thromboxane and Leukotriene Research, pp. 47. Billah, M. M., and Anthe, J. C. The regulation and cellular functions of 590-593. New York: Raven Press. 1989. phosphatidylcholine hydrolysis. Biochem. J., 269: 281-291. 1990. 28. Smith, M. R., Liu, Y-L., Kim, H., Rhee, S. G., and Kung, H. F. Inhibition 48 Larrodera, P., Cornet, M. E., Diaz-Meco, M. T., Lopez-Barahona, M., Diaz- of serum- and nu-stimulated DNA synthesis by antibodies to phospholipase Laviada, I.. Guddal, P. H., Johansen, T.. and Moscai. J. Phospholipase C- C Science (Washington DC), 247: 1074-1077, 1990. mediated hydrolysis of phosphatidylcholine is an important step in PDGF- 29. Müller-Decker,K., Doppier, C., Amtmann, E., and Sauer, G. Interruption stimulated DNA synthesis. Cell, 61: 1113-1120, 1990. of growth signal transduction by an antiviral and antitumoral xanthate 49. Storme, G. A., Berdel, W. E., van Blitterswijk. W. J., Bruyneel. E. A., De compound. Exp. Cell Res., 177: 295-302, 1988. Bruyne, G. K., and Mareel, M. M. Anti-invasive effect of racemic l-O- 30. Amtmann, E., and Sauer. G. Selective killing of tumor cells by xanthates. octadecyl-2-O-methylglycero-3-phosphocholineon M04 mouse fibrosarcoma Cancer Lett., 35: 237-244, 1987. cells in vitro. Cancer Res., 45: 351-357, 1985. 31. Prentki, M., Deeney, J. T., Matschinsky, F. M., and Jospeh, S. K. Neomycin: 50. Noseda, A., Godwin, P.L., and Modest, E. J. Effects of antineoplastic ether a specific drug to study the inositol-phospholipid signalling system? FEBS lipids on model and biological membranes. Biochim. Biophys. Acta, 945: Lett., /97: 285-288, 1986. 92-100, 1988. 32. van Blitterswijk, W. J., Hilkmann, H., and Storme, G. A. Accumulation of 51. Daniel, L. W., Small, G. W., and Strum, J. C. Characterization of cells an alkyl-phospholipid in tumor cell membranes affects membrane fluidity sensitive and resistant to ET-18-OCH,. Proc. Am. Assoc. Cancer Res., 31: and tumor cell invasion. Lipids. 22: 820-823, 1987. 412. 1990. 33. Martin, T. W. Receptor regulation of phosphoinositidase C. Biochim. Bio- 52. Bazill, G. W., and Dexter, T. M. Role of endocytosis in the action of ether phys. Acta, 962: 282-296, 1991. lipids on WEHI-3B, HL60. and FDCP-mix A4 cells. Cancer Res., 50:7505- 34. Meisenhelder, J., Suh, P-G., Rhee, S. G., and Hunter, T. Phospholipase C- 7512, 1990. y is a substrate for the PDGF and EGF receptor protein-tyrosine kinoses in 53. Chabot, M. C., Wykle, R. L., Modest, E. J., and Daniel, L. W. Correlation vivo and in vitro. Cell, 57: 1109-1122, 1989. of ether lipid content of human leukemia cell lines and their susceptibility to 35. Wahl, M. I., Oleshaw, N. E., Nishibe, B., Rhee, S. G., Pledger, W. J., and 1-O-octadecyl^-O-methyl-roc-glycero-S-phosphocholine. Cancer Res., 49: Carpenter, G. Platelet-derived growth factor induces rapid and sustained 4441-4445. 1989. tyrosine phosphorylation of phospholipase C-y in quiescence BALB/c 3T3 cells. Mol. Cell. Biol., 9: 2934-2943, 1989. 54. Diomede, L., Bizzi, A., Magistrelli. A.. Modest, E. J., Salmona, M., and Noseda, A. Role of cell cholesterol in modulating antineoplastic ether lipid 36. Bigay, J., Deterre, P., Pfister. C., and Chabre, M. Fluoride complexes of uptake, membrane effects and cytotoxicity. Int. J. Cancer, 46: 341-346, aluminium or beryllium act on G-proteins as reversibly bound analogues of the gamma phosphate of GTP. EMBO J., 6: 2907-2915, 1987. 1990. 55. Smith. M. R., Ryu, S. H., Suh, P. G., Rhee, S. G., and Kung, H. F. S-phase 37. Taylor, S. J., Chae, H. Z., Rhee, S. G., and Exton, J. H. Activation of the 01 isozyme of phospholipase C by n subunits of the <.,, class of G proteins. induction and transformation of quiescent NIH 3T3 cells by microinjection Nature (Lond.), 350: 516-518, 1991. of phospholipase C. Proc. Nati. Acad. Sci. USA, 86: 3659-3663, 1989. 38. Goldschmidt-Clermont, P. J., Kim, J. W., Machesky, L. M., Rhee, S. G., 56. Rath, H. M., Doyle, G. A. R., and Silbert, D. F. Hamster fibroblasts defective and Pollard, T. D. Regulation of phospholipase C-gammal by proli Iin and in thrombin-induced mitogenesis. J. Biol. Chem., 264:13387-13390, 1989. tyrosine phosphorylation. Science (Washington DC), 251: 1231-1233, 1991. 57 Margolis, B., Zilberstein, A., Franks, C., Felder, S., Kremer, S., Ullrich. A., Rhee, S. G., Skorecki, K., and Schlessinger, J. Effect of phospholipase C--y 39. Carter, H. R., Wallace, M. A., and Fain, J. N. Purification and characteriza tion of l'I ('.;,„.a muscarinic cholinergic regulated phospholipase C from overexpression on PDGF-induced second messengers and mitogenesis. Sci rabbit brain membrane. Biochim. Biophys. Acta, 1054: 119-128. 1990. ence (Washington DC), 24«:607-616. 1990. 40. Majerus, P. W., Ross, T. S., Cunningham. T. W., Caldwell, K. K., Jefferson. 58 Cuadrado, A., and Molloy, C. J. Overexpression of phospholipase C-y in A. B., and Bansal, V. S. Recent insights in phosphatidylinositol signaling. NIH 3T3 fibroblasts results in increased phosphatidylinositol hydrolysis in Cell, 63:459-465, 1990. response to platelet-derived growth factor and basic fibroblast growth factor. 41. Perrella, F. W., Piantadosi, C., Marasco, C. J., and Modest, E. J. Inhibition Mol. Cell. Biol., 10: 6069-6072, 1990. of phospholipase C and cell growth by ether lipid analogues of phosphati 59. Helfman, D. M., Barnes. K. C.. Kinkade, J. M., Vogler, W. R., Shoji. M. M., and Kuo, J. F. Phospholipid-sensitive Ca2*-dependent protein phos dylinositol. Proc. Am. Assoc. Cancer Res.. 31:409. 1990. 42. Berridge, M. J. Rapid accumulation of inositol trisphosphate reveals that phorylation system in various types of leukemic cells from human patients agonists hydrolyse polyphosphoinositides instead of phosphatidylinositol. and in human leukemic cell lines HL60 and K562. and its inhibition by alkyl- Biochem. J., 272: 849-858, 1983. phospholipid. Cancer Res.. 43: 2955-2961, 1983.

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G. Powis, M. J. Seewald, C. Gratas, et al.

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