Effects of Thioether Phospholipid BM 41.440 on Protein Kinase C and Phorbol Ester-Induced Differentiation of Human Leukemia HL60 and KG-1 Cells1

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[CANCER RESEARCH 48, 6669-6673, December 1, 1988] Effects of Thioether Phospholipid BM 41.440 on Protein Kinase C and Phorbol Ester-induced Differentiation of Human Leukemia HL60 and KG-1 Cells1

Mamoru Shoji, Robert L. Raynor, Wolfgang E. Berdel,2 William R. Vogler, and J. F. Kuo

Departments of Medicine (Hematology/Oncology) [M. S., W. E. B., W. R. V.] and Pharmacology [R. L. R., J. F. K.J, Emory University School of Medicine, Atlanta, Georgia 30322

ABSTRACT possible effects of BM 41.440 on the enzyme system. We found that the thioether was a rather potent and specific PKC inhib The synthetic thioether phospholipid BM 41.440 (l-5-hexadecyl-2- methoxymethyl-rac-glycero-3-phosphocholine) was found to inhibit pro itor, which could be useful in examining cellular processes in tein kinase C (PKC) activity competitively with respect to phosphatidyl- which the enzyme system is likely to participate. serine, with an apparent AÂ¡valueof about 6.4 Â¿tM.Theagent also inhibited the enzyme activated by diolein or 12-0-tetradecanoylphorbol-13-acetate MATERIALS AND METHODS (TPA), without affecting binding of [3H]phorbol-12,13-dibutyrate to the enzyme. Myosin light chain kinase and cAMP-dependent protein kinase Materials. Medium and chemicals used for cell culture were obtained were not inhibited by BM 41.440, indicating a specificity of the action of from GIBCO (Grand Island, NY); TPA and PDBu were from LC the agent. BM 41.440 partly blocked the TPA-induced depletion of Service (Woburn, MA); DMSO was from Fisher Scientific Co. (Atlanta, soluble PKC in HL60 and KG-1 cells (responsive to the differentiating GA); diolein, PS (bovine brain), lyso-PC, ouabain, RA (all-ira/w), and effect of TPA) but not in K562 cells (resistant to the TPA effect). The histone HI (type HI-S) were from Sigma Chemical Co. (St. Louis, thioether inhibited the phosphatidylserine/Ca^-dependent phosphoryla- MO); VD3 was from Dr. M. R. Uskokovic, Hoffman-LaRoche, Inc. tion of several common proteins in the solubilized homogenates of 111.60 (Nutley, NJ); ET-18-OCHj was from Medmark Chemicals (Gruenwald and KG-1 cells, and that of apparently distinct proteins in the preparation bei Muenchen, FRG); BM 41.400 was from Boehringer Mannheim of K562 cells. The TPA-induced differentiation of 111.60and KG-1 cells (Mannheim, FRG); [3H]PDBu (15.8 Ci/mmol) was from New England was inhibited by BM 41.440 at a concentration inhibitory to PKC, but Nuclear (Boston, MA). differentiation of 1116(1 cells promoted by dimethyl sulfoxide, retinole Cell Culture and Cell Differentiation. HL60, KG-1, and KS62 cells acid, and 1,25-dihydroxyvitamin l)i, on the other hand, was not affected. were cultured in RPM1 1640 medium supplemented with penicillin G The present data suggested that PKC inhibition might partly account for (10 units/ml), streptomycin sulfate (10 Â¿ig/ml),and L-glutamate (0.3 the antineoplastic effect of BM 41.440, and that the agent could be useful mg/ml) and either 20% (for KG-1) or 10% (for HL60 and K562) heat- inactivated fetal calf serum, in a humidified incubator at 37Â°Cin 95% in studying involvements of the PKC system in cellular processes. air/5% CO:. The cells at the mid-log growth phase were harvested and used in all experiments reported herein. The TPA-induced differentia INTRODUCTION tion of HL60 and KG-1 cells via the macrophage pathway were deter The ALP3 derivative ET-18-OCH3 has been shown to possess mined by cell adhesion after treatments of the cells for 2 days with TPA and other agents, as described previously (13) and in Table 1. antitumor activity, because it is cytotoxic to leukemia (1,2) and Differentiation of HL60 cells to matured granulocytes/monocytes after tumor cells (3) in vitro, inhibitory to the growth (4) and metas treatment for 7 days with DMSO, RA, or VDj was determined by the tasis (5) of syngeneic murine tumors, and effective in the nitroblue tetrazolium reduction method of Feigin et al. (14) modified treatment of experimental rat tumors (6). Various cellular and recently by Zylber-Katz and Glazer (15). molecular mechanisms underlying the actions of the ether lipid PKC Preparations and Assay and Endogenous Protein Phosphoryla- have been suggested, including enhancement of cytotoxicity of tion. PKC was partially purified (about 20-40% homogenous) from pig macrophages (5), and inhibition of phospholipid metabolism brain extracts through the phenyl Sepharose step (16) or purified to (7) and PKC activity (8). ET-18-OCH3 also inhibits phosphor- homogeneity from rat brain extracts through the polylysine step (17). ylation of various endogenous proteins and differentiation of PKC was assayed under the standard conditions as described elsewhere (13, 18). Briefly, the reaction mixtures (0.2 ml) contained 5 fimol of HL60 cells promoted by TPA (9), strongly suggesting that piperazine-jV,yV'-bis(2-ethanesuIfonicacid) (pH 6.5), 2 ^mol of MgClz, inhibition of PKC-dependent reactions may represent a basis 5 Mgof PS, 40 ug of histone H l, 0.06 ^mol of EGTA, with or without for the observed ALP effects. More recently, a number of TLP 0.1 Mmol of CaCl2, 1.0 nmol of [r-32P]ATP (containing about 1 x IO6 deviatives (the thioether analogs of ALP) have been synthesized cpm), and various kinds and concentrations of agents, as indicated. and tested as potential antineoplastic agents. One of which, When present, the final concentration of CaCl2 was 200 uM (in excess BM 41.440, has been found to be effective against various of 300 MMEGTA). The reaction was carried out at 30Â°Cfor 5 min. In leukemia cells and solid tumors (10, 11). Because of a central experiments (Fig. 4) that examined the specific stimulation of PKC role played by PKC in cellular regulation (12), we investigated activity by TPA or diolein and its inhibition by BM 41.440, the enzyme was assayed under the modified conditions using low concentrations of Received 4/22/88; revised 8/19/88; accepted 8/26/88. PS (2 Mg/0.2 ml assay volume) and CaCI2 (10 ^M), as reported recently The costs of publication of this article were defrayed in part by the payment (18, 19). Phosphorylation of endogenous proteins in the total solubi of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. lized fraction (homogenate) of leukemic cells was carried out as de 1Supported by USPHS Research Grants CA-29850 (W. R. V.) and CA-36777, scribed previously (8, 13) and as indicated in Fig. 7. HL-15696, and NS-17608 (J. F. K.). Other Methods. [3H]PDBu binding to PKC was carried out as de 2 Permanent address: Division of Hematology and Oncology, Department of scribed (18-20) and as indicated in Fig. 5. MLCK was purified from Medicine l. Technische UniversitÃ¤t,Munich, Federal Republic of Germany. 3The abbreviations used are: ALP, alkyllysophospholipid; TLP, thioether rabbit skeletal muscle (21) and A-PK was purified from pig heart (22); lysophospholipid; BM 41.440, l-S-hexadecyl-I-methoxymethyl-rac-glycero-S- their activities were assayed as described (21, 22). [r-32P]ATP was phosphocholine; ET-18-OCH3, 1-O-octadecyl^-O-methyl-rac-glycero-S-phos- prepared by the method of Post and Sen (23). phocholine; PKC, protein kinase C; MLCK, myosin light chain kinase; A-PK, cAMP-dependent protein kinase; TPA, 12-0-tetradecanoylphorbol-13-acetate; PDBu, phorbol-12,13-dibutyrate; DM SO. dimethyl sulfoxide; RA, retinole acid (Â»II(rum): VDj, 1,25-dihydroxyvitamin D3; PS, phosphatidylserine; PC, phos- RESULTS phatidylcholine; NBT, nitroblue tetrazolium; EGTA, ethyleneglycol bis(0-ami- noethylether)-/VyV,A",JV"-tetraacetic acid; ICso, concentration causing 50% inhi The structures of the ALP prototype ET-18-OCH3 and the bition. TLP prototype BM 41.440 are shown below (Fig. 1). BM 6669 Downloaded from cancerres.aacrjournals.org on September 24, 2021. © 1988 American Association for Cancer Research. INHIBITION OF PKC SYSTEM BY THIOETHER LIPID Table I Effects ofBM 41.440 on HL60 and KG-1 cell differentiation those made previously for ET-18-O-CH3 (8). BM 41.440, how Cells were incubated for l h with or without 5 Â«iMBM41.440, washed once ever, inhibited PKC noncompetitively with respect to Ca2+, with and cultured (2 days) in fresh medium in the presence or absence of 10 n\i TPA. The differentiating cells were indicated by adhesion to culture dishes. with an apparent K, of 22.2 pM (data not shown). The value was higher than that seen in Fig. 3 because the enzyme was differentiation viability assayed in the presence of a saturating concentration (25 /ig/ CelllineHL60 (% oftotal)2.1 (% oftotal)90 ml) of PS as in Fig. 2, consistent with that data shown in Fig. (control) Â±0.2 +92 3 that BM 41.440 and PS were likely interacting with function TPA 45.4 Â±2.4Â° Â±:84 BM 4 1.440 2.6 Â±0.4 Â± ally similar site(s) on PKC. BM 4 1.440 + TPA 30.9 Â±2.0"-* 96 Â± BM 41.440 inhibited PKC assayed under a suboptimal con KG-1TreatmentNoneNone (control) 1.0 Â±0.2 93 Â±. TPA 25.8 Â±0.8 98 Â± dition (i.e., using a low PS concentration of 10 Â¿ig/mland a low BM 4 1.440 1.2 Â±0.3 96 Â± CaCl2 concentration of 10 /Ã•M),withan estimated IC5oof about 19.4 Â±0.5"' *Cell BM 4 1.440 + TPACell 97 +[i; 10 UM(Fig. 4). The agent also inhibited the enzyme maximally â€¢¿Significantlydifferent from the control (mean Â±SE, P < 0.001; N = 3). * Significantly different from the value seen with TPA alone (P < 0.05; N â€¢¿â€¢activated by diolein or TPA, but with much higher ICso values of 50 and 90 Â¿Â¿M,respectively(Fig. 4). The data appeared to 3). suggest that the PKC activators (diolein and TPA) antagonized H2 C-O-(CH2J17-CH3 H2 C-S-(CH2)15-CH3 the action of the PKC inhibitor (BM 41.440) by directly inter acting at the common binding site(s) on PKC or PKC-PS-Ca2* HC-O-CH3 HC-CH2-O-CH3 ternary complex. This notion, however, was not supported by the [3H]PDBu binding experiments (Fig. 5). Although diolein | O O H2 C-0-P-0-

ET-18-OCH, BM 41.440

Fig. 1. Structures of ET-18-OCHj and BM 41.440.

100

> 40 O PKC(ptenyl O â€¢¿PKC(polylyi.nÂ») A HL60 (crude Â«Urici) O 20 A RAT BRAM (crwd* â€¢¿â€¢Traci)

10 20 30 100 0 BM 41.440 (uM) 0 10 20 30 40 50 Fig. 2. Comparative effects of BM 41.440 on PKC, MLCK, and A-PK. The PHOSPHATIDYLSERINE (ug/ml) enzymes were assayed under the standard conditions in the presence of various concentrations of BM 4 1.440, as indicated. The PS/Ca2*-stimulated PKC activity Fig. 3. Inhibition of PKC by BM 41.440 as a function of PS concentrations. The enzyme as assayed under the standard conditions except for various concen (piiicil min) for the pig or rat brain enzyme from the steps of phenyl Sepharose trations of PS and the agent, as indicated. The double-reciprocal plots of the data (12.2) and the polylysine (9.8), and the crude extracts from rat brains (8.9) and HL60 cells (6.5) were respectively taken as 100%. The activity (pmol/min) of are also shown (inset). Similar results were obtained in two other experiments. MLCK stimulated by calmodulin/Ca2* (12.5) and A-PK stimulated by cAMP (10.2) were also taken as 100%, respectively. Similar results were obtained in 24 three other experiments. 20 41.440 inhibited four PKC preparations with comparable ICso values of 15-20 fiM (Fig. 2). The preparations included crude 16 extracts from HL60 cells and rat brains, and a highly purified (from the phenyl Sepharose step) and a homogenous (from the 12 polylysine step) enzyme obtained from the soluble fraction of pig or rat brains. BM 41.440, however, was without effect on MLCK (a calmodulin/Ca2+-dependent enzyme) and A-PK, in dicating that the thioether, as ET-18-OCH3 (8) and certain polypeptides such as defesins (18), was a specific PKC inhibitor. Because of the limited supply of the homogeneous enzyme and IIâ€”o the demonstrated similarity of the inhibitory action of the agent 20 40 60 80 100 200 irrespective of the enzyme purity, we employed the enzyme BM 41.440 (uM) from the phenyl Sepharose step for all other appropriate studies Fig. 4. Inhibition of diolein- or TPA-stimulated PKC activity by BM 41.440. reported herein. The enzyme was assayed under the modified conditions using suboptimal concen Kinetic analysis indicated that BM 41.440 inhibited PKC trations of PS and CaCl j but in the presence or absence of diolein (l /IM) or TPA competitively with respect to PS, with an average apparent K-, (20 IIM).as indicated. Horizontai bars, activity levels seen in the absence of CaCl2 (i.e., in the presence of 10 mm EGTA) or the PKC activators. Similar results value of 6.4 pM (Fig. 3), the findings which were similar to were confirmed in another set of experiments. 6670 Downloaded from cancerres.aacrjournals.org on September 24, 2021. © 1988 American Association for Cancer Research. INHIBITION OF PKC SYSTEM BY THIOETHER LIPID

120 100Â£ A. HL60 B. HL60

o 100 s C o â€¢¿580u

o 60 o m 40 m3 o 20

20 40 60 80 200 AGENT (uM) Fig. 5. Comparative effects of ET-18-OCH3, BM 41.440, and diolein on the binding of [3H]PDBu to PKC. The enzyme (5 lipids were also unable to substitute for PS to support the phorbol ester binding to the enzyme (data not shown). We reported recently that TPA caused PKC translocation from cytoplasm to plasma membrane in KG-1 (13) and HL60 cells (24), an initial step presumably essential for cell adhesion and differentiation of the two cell lines in response to TPA. It o 10 30 100 o 10 100 was found in the present studies that BM 41.440 partly blocked translocation of soluble PKC in HL60 (Fig. 6B) and KG-1 cells TPA (nM) (Fig. 6Q, as did ET-18-OCH3 in HL60 cells (Fig. 6A). BM Fig. 6. Effects of ET-18-OCH 3and BM 41.440 on the TPA-induced depletion 41.440, however, had no effect on the TPA-induced depletion of PKC in HL60, KG-1, and K562 cells. The cells were incubated with or without 20 JIMET-18-OCHj or BM 41.440 for 1 h, followed by incubations with various of soluble PKC in K562 cells (Fig. 60), a cell line that is concentrations of TPA for 10 min, as indicated. The enzyme activity in the soluble unresponsive to the differentiating effect of TPA. It should be fraction (1-4 /ig protein) was assayed under the standard conditions. The enzyme activity levels seen with cells treated without TPA were taken as 100% in all noted here that TPA caused an initial redistribution of cyto- cases. See "Materials and Methods" for further details. Similar results were plasmic PKC to the nuclear region in K562 cells and TPA- obtained in two or three other experiments. *, significantly different from the unresponsive fibroblastic cell lines (25) rather than to the control (P < 0.05; n = 3). plasma membrane in HL60 (24, 25) and KG-1 cells (13). DISCUSSION There were a number of apparently similar substrate proteins (M, 16,000-81,OOQ) for PKC (i.e., PS/Ca2+-dependent phos- We reported previously that ET-18-OCH3 (ALP), an anti- phorylation) in the solubilized homogenates of HL60 and KG- neoplastic ether lipid, inhibited PKC activity (8) and blocked 1 cells and, furthermore, phosphorylation of these proteins TPA-dependent phosphorylation of endogenous proteins in were inhibited, in a dose dependent manner, by BM 41.440 HL60 cells and TPA-induced differentiation of the leukemic (Fig. 7). Phosphorylation of PKC substrate proteins (M, cells (9). In the present studies, we found that BM 41.440 20,000-81,000) from K562 cells, distinct from those seen for (TLP), a structural analog of ALP, also inhibited PKC activity HL60 and KG-1 cells, was similarly inhibited by the agent (Fig. and TPA-induced differentiation of HL60 and KG-1 cells. 7). We reported previously inhibition of phosphorylation of Although the molecular mechanisms underlying the biological PKC substrate proteins by ET-18-OCH3 in these cell lines (8, effects of BM 41.440 are still unknown, PKC inhibition could be a critical factor, particularly in view of a pivotal role played 9). TPA-induced differentiation of HL60 (24,26) and KG-1 cells by this key phosphorylation system in biological processes including transmembranal signaling and cell growth and differ (13, 27) has been shown to be PKC-dependent, the contention entiation (12). also substantiated by the findings that the PKC inhibitor ET- BM 41.440 inhibited PKC competitively with respect to PS 18-OCH3 antagonized the differentiating effect of TPA on but noncompetitively with respect to Ca2+, indicating that the HL60 cells (9). Consistent with the above data were the present thioether phospholipid, as ET-18-OCH3 reported previously findings that BM 41.440, devoid of cytotoxicity at the concen (8), interacted with site(s) on PKC also shared by PS but not tration (5 /Â¿M)used, also partially blocked differentiation of by Ca2+. It appeared that BM 41.440 did not bind to site(s) both cell lines induced by TPA (Table 1). Differentiation of shared by diacylglycerol or TPA, because they had no effect on HL60 cells promoted by DMSO, RA, or VD3, the process likely [3H]PDBu binding to the PKC/PS/Ca2+ ternary complex. In to be PKC-independent, on the other hand, was unaffected by teraction of BM 41.440 with the complex, however, would the PKC inhibitor (data not shown). affect the action of the PKC activators, because the enzyme 6671 Downloaded from cancerres.aacrjournals.org on September 24, 2021. © 1988 American Association for Cancer Research. INHIBITION OF PKC SYSTEM BY THIOETHER LIPID

B. KG-1 A. HL60 C. K562 23456 123456 123456769

-61K Fig. 7. Inhibition by BM 41.440 of the PS/ Ca2*-dependent phosphorylation of endoge -Â«5K nous proteins from HL60, KG-1, and K562 I -53K cells. The total solubilized homogenates of the -46K cells (500 mg protein/ml) was phosphorylated in the presence or absence of CaCh (500 ^M), -40K -36K PS (25 Mg/ml) and various concentrations of ' -34K BM 41.440. as indicated. See "Materials and I

Methods" for further details. Similar results -27K were obtained in two other experiments. -22K -20K

CaCI2 -

PS +++++ + + BM41440 â€”¿â€”¿ â€”¿â€”¿13 25 50100200 â€”¿â€”¿1325 50 100 200 â€”¿â€”¿â€”¿â€”¿ 13 25 50 100200

maximally activated by TPA or diolein was also inhibited by aid of specific PKC inhibitors such as BM 41.440, would give the agent. We observed recently that the branched-chain ana new insights into this basic biological phenomenon. logs of distearoyl PC could either activate or inhibit the TPA- stimulated PKC without affecting the [3H]PDBu binding (19). ACKNOWLEDGMENTS In this respect, it is of interest that of the two peptide PKC inhibitors of similar potency, polymyxin B could whereas de- We thank Dr. James T. Stull for the preparation of MLCK and Rebecca Brooks for skillful preparation of the manuscript. fensin could not inhibit the phorbol ester binding (18), suggest ing a critical structural requirement for the PKC inhibitors to block the binding of PKC activators. These observations, when REFERENCES taken together, also suggested that binding of phorbol ester or 1. Andreesen, R., Modolell, M., Weltzien, H. U., Eibl, H., Common, H. H., diacylglycerol to the PKC/PS/Ca2+ complex might not be nec Loehr, G. W., and Munder, P. G. Selective destruction of human leukemic cells by alkyl-lysophospholipids. Cancer Res., 3Â«:3894-3899, 1978. essarily correlated with the activation state of the enzyme. The 2. Tidwell, T., Guzman, G., and Vogler, W. R. The effect of alkyllysophospho- apparent contradiction that the PKC inhibitor BM 41.440 could lipids on leukemic cell lines. I. Differential action on two human leukemic counteract the TPA-induced PKC translocation and cell differ cell lines HL60 and K562. Blood, 57: 794-797, 1981. 3. Berdel, W. E., Fink, U., Egger, B., Reichert, A., Munder, P. G., and Rasetter, entiation without affecting PDBu binding to PKC is intriguing. J. Inhibition by alkyl-lysophospholipids of tritiated thymidine uptake in cells Although the underlying mechanisms are unclear, it is likely of human malignant urologie tumors. J. Nati. 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Mamoru Shoji, Robert L. Raynor, Wolfgang E. Berdel, et al.

Cancer Res 1988;48:6669-6673.

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