Comp. Biochem. PhysioL Vol. 97B, No. 1, pp. 151-158, 1990 Printed in Great Britain

0305-0491/90 $3.00+ 0.00 © 1990 PergamonPress plc

INHIBITION OF INTRINSIC PROTEIN TYROSINE KINASE ACTIVITY OF EGF-RECEPTOR KINASE COMPLEX FROM H U M A N BREAST CANCER CELLS BY THE MARINE SPONGE METABOLITE (+)-AEROPLYSININ- 1 MATTHIAS-H. KREUTER,* ROBIN E. LEAKE,t FRANK RINALDI,~" WOLFGANGMOLLER-KLIESER,:~ARMIN MAIDHOF,* WERNER E. G. Mf2LLER* and HEINZ C. SCHRODER*§ *Institut for Physiologische Chemie, Abteilung Angewandte Molekularbiologie, Universit/it, Duesbergweg 6, 6500 Mainz, FRG (Tel: 061 31 395919); tDepartment of Biochemistry, University of Glasgow, Glasgow G12 8QQ, UK; and :l:Institut fiir Physiologie und Pathophysiologie, Universit/it, Duesbergweg 6, 6500 Mainz, FRG (Received 29 January 1990)

(+)-Aeroplysinin-l, a naturally occurring tyrosine metabolite from the marine sponge Verongia aerophoba, was found to inhibit the phosphorylation of lipocortin-like proteins by a highly purified preparation of the epidermal growth factor (EGF) receptor-tyrosine protein kinase complex from MCF-7 breast carcinoma cells. 2. (+)-Aeroplysinin-1 blocked the EGF-dependent proliferation of both MCF-7 and ZR-75-! human breast cancer cells and inhibited the ligand-induced endocytosis of the EGF receptor in vitro. 3. Treatment with aeroplysinin-I in the concentration range at 0.254).5 #M resulted in a time- and dose-dependent total tumor cell death in vitro. 4. At a 10-fold higher concentration the compound did not reveal any cytostatic activity in normal human fibroblasts. 5. From these data we conclude that (+)-aeroplysinin-1 represents a compound which displays a strong anti-tumor effect on EGF-dependent tumor cell lines. Abstract--l.

dependent protein-tyrosine kinase activity (for a review, see Carpenter, 1987; Yarden and Ullrich, (+)-Aeroplysinin- 1, an optically active 1,2-dihy- 1988). The tyrosine kinase activity of the E G F recepdroarene-l,2-diol with the chemical structure of 1- tor is assumed to be required for EGF-induced effects (3',5'-dibromo-l',2'-dihydroxy-4'-methoxycyclohexa- on gene transcription, calcium mobilization, receptor 3',5'-dien-l'-yl) methylcyanide (Fig. 1) is a naturally down-regulation and stimulation of cell growth occurring tyrosine metabolite from the marine (Cben et al., 1987). sponge Verongia aerophoba (Fattorusso et al., 1970, Here we show that (+)-aeroplysinin-1, which pos1971; Fulmor et al., 1970). In the course of a screen- sesses a close structure-relationship to tyrosine, ing program of a number of bioactive substances blocks the EGF-dependent proliferation of both isolated from marine sponges we observed that MCF-7 and ZR-75-1 breast cancer cells and inhibits L5178y mouse lymphoma cells are much more sensi- the ligand-induced endocytosis of the E G F receptor tive than spleen lymphocytes against this compound in ZR-75-1 cells in vitro. (+)-Aeroplysinin-I was (Kreuter et al., 1989). Testing a series of cell lines we found to inhibit the tyrosine-specific phosphorylation ascertained human epithelial and epithelial-like of lipocortin-like proteins, which have been estabtumor cells (MCF-7 breast cancer cells, and WiDr lished as major substrates of the E G F receptorcolon carcinoma cells) to be most sensitive to treat- associated protein-tyrosine kinase (Pepinsky and ment with (+)-aeroplysinin-1. A common property Sinclair, 1986), by a highly purified preparation of of these cell lines is a marked dependence on growth the E G F receptor protein-tyrosine kinase complex, factors (Engel and Young, 1978; Osborne et al., isolated from MCF-7 cells. 1982, 1987). The epidermal growth factor (EGF) receptor being present at the surface of these cells has been shown to be a transmembrane glycoprotein of MATERIALS AND METHODS M, 170,000 which possesses an intrinsic, ligandMaterials §Author to whom correspondence should be addressed. The following materials were obtained: murine [12sI]Abbreviations used--DMSO, dimethylsulfoxide; EGF, epi- epidermal growth factor ([12~I]EGF;spec. act., 170 #Ci/#g) dermal growth factor; Hepes, 4-(2-hydroxylethyl)-land [6:H]thymidine (dThd) (15Ci/mmol) from New piperazineethanesulfonic acid; PBS, phosphate-buffered England Nuclear (Boston, MA, USA); and [~:2p]ATP saline; PMSF, phenylmethanesulfonyl fluoride; SDS, (3000 Ci/mmol) from Amersham International (Buckingsodium dodecyl sulfate. hamshire, UK). INTRODUCTION

151

152

MATTHIAS-H. KREUTERet al. OCH3

NO CH2CN Fig. 1. Structural formula of (+)-aeroplysinin-1.

was used. One milliliter aliquots of a ZR-75-1 cell suspension (1.1-1.45 x 105 cells/ml) were added to 24-well tissue culture plates. The cells were allowed to plate down overnight in an unhumidified incubator (37°C; 5% CO,). (+)-Aeroplysinin-1 (0.34-4.8/~M) was added to sets of 12 wells/each concentration. After incubation for 24 hr, each set of 12 wells was assessed for EGF binding capacity and DNA content. Determination of DNA

(+)-Aeroplysinin-1 (mol. wt 339.6; Fulmor et al., 1970) was isolated from the sponge Verongia aerophoba, which was collected in the bay of Kotor (Yugoslavia), as described previously (Kreuter et al., 1989). The antiserum used was raised in rabbits against calelectrin from Torpedo marmorata (Fritsche et al., 1988) and was a kind gift from Dr U. Fritsche (Max-Planck-Institut fiir biophysikalische Chemie, Abteilung Neurochemie, G6ttingen, FRG). The anti-calelectrin antibodies were purified by affinity chromatography on protein A-Sepharose (Ey et al., 1978); they cross-reacted with proteins of M, 68,000, 34,000 and 32,000 in rat brain (Stoll et al., 1988). Cell culture Human mammary carcinoma cells (MCF-7) (Engel and Young, 1978; Osborne et al., 1987) were grown in Eagle's basal medium supplemented with 10% fetal calf serum at 37°C in 5% CO2 and air. ZR-75-1 breast cancer cells (Engel and Young, 1978; Engel et al., 1978) were grown in Dulbecco's modified Eagle's medium (GIBCO) in plastic flasks supplemented with 10% newborn calf serum at 37°C in 5% C02 and air. Measurement o f EGF-mediated cell proliferation EGF-dependent proliferation of MCF-7 cells was determined using 96-well microtiter plates containing 4000 cells/well, suspended in 200/al of serum free Eagle's basal medium. The cells were allowed to plate down for 24 hr. For the experiments the wells were washed twice with 200/~1 of phosphate-buffered saline (PBS), followed by basal medium supplemented with 5% newborn calf serum or 10% fetal calf serum. In a further set of experiments the synchronized ceils were additionally treated with 0.05-0.2 # M ( +)-aeroplysinin-I (dissolved in DMSO). The final DMSO concentration did not succeed 0.05%; at this concentration DMSO was determined to have no influence on the experiment. Twelve hours prior to the end of incubation 0.1/~Ci of [3H]dThd/ml (=6.7nM) was added. Incorporation of [3H]dThd was determined as described (Mfiller et al., 1983). EGF receptor binding assay After washing twice with 1 ml of PBS, 100/~1 of [125I]EGF (0.4-0.6 ng) in Dulbecco's minimum essential medium containing l mg/ml of bovine serum albumin or 100/tl of [1251]EGF containing a 250-fold excess of unlabeled EGF were added per well. This allows for the measurement of both total binding and non-specific binding. The wells were then incubated for 30 min at 37°C in the presence of 5% CO,. At the end of the incubation period, the wells were washed twice with PBS. Then 100/d of an SDS mix (2% SDS, 4mM EDTA, 10% butanol, 6% 4-aminosalicylic acid) were added to each well, and extraction was performed for 5rain at room temperature. The extract was then counted for radioactivity in a gamma scintillation counter. The binding data were evaluated according to Scatchard using the LIGAND program (Munson and Rodbard, 1980), Measurement o f EGF receptor accumulation An indirect method combining the measurement of the amount of bound [mI]EGF and of DNA synthesis in the presence of different concentrations of (+)-aeroplysinin-1

After washing twice with PBS, I00/~1 of ETN buffer (10 mM EDTA, 10 mM Tris-HC1, 100 mM NaCI; pH 7.0) containing 0.2% SDS were added to each well, and the plates were incubated at 37°C for 15 min in an unhumidified incubator in 5% CO2/air. After solubilization (checked by phase contrast microscopy) the content of each well was transferred to a separate tube and 2.4 ml of ETN buffer containing 100ng/ml Hoechst 33258 and 5#g/ml RNase were added. The tubes were incubated for 15 min at 37c'C in the dark, and the fluorescent enhancement was measured at 360 nm using a Hitachi Perkin Elmer Fluorescent spectrophotometer. The viability of the cells was determined by Trypan Blue exclusion (0.5% w/v). Preparation and solubilization o f membrane vesicles from MCF-7 breast cancer cells Twenty-four hours prior to starting shed membrane vesicle preparation from MCF-7 cells, fresh medium without serum was added to the cells. One hour prior to starting the preparation 100ng/ml (final concentration) of EGF (from mouse submaxillary gland; Sigma) was added. Ten 100-ml flask bottles of confluent cells were used for the experiment. The cells were washed three times with 50 ml of Dulbecco's phosphate-buffered saline at room temperature and subsequently rinsed with I0 ml/flask of ice-cold hypotonic buffer (10ram Hepes pH 7.5, 0.5 mM MgCI2). Subsequently the cells were centrifuged (3000 g, 5 min) and the supernatant was discarded. The pellets were resuspended in 5 ml of vesiculation buffer (50 mM sodium phosphate pH 8.5, 100 mM NaC1, 5 mM KCI, 10 mM EDTA) (Cohen et al., 1982; with modifications), allowed to stand on ice for 10 rain, sonicated for 10 sec (Brown sonifier, at setting 4) and additionally incubated on ice for 10 rain. Then 20 mM CaC12 were added and the suspension was centrifuged (2000 g, 5 rain) to remove debris. The resulting supernatant, containing vesicularious membrane particles, similar to the described shed membrane vesicles (Cohen et al., 1982), was centrifuged at 20,000g for 15rain and the membraneous pellet was resuspended in 5 ml of membrane solubilization buffer (20raM Hepes pH7.4, 1% Triton X-100, 10% glycerol) (according to Cohen et al., 1980, 1982; with modification). After incubation for 20 rain at room temperature, the suspension was supplemented with 7 ml of Ca 2+- and Mg2+-free PBS, containing 10 mM EDTA, and centrifuged at 20,000g for 15 rain. The supernatant was concentrated, using an UltraFree centrifugation cup (NMWL 30 KD; Millipore), at 2000g for 30 rain at 4~'C. The concentrated fraction was suspended in PBS and then centrifuged at 20,000g for 15 rain at 4°C. The supernatant was concentrated again after addition of 2 mM dithiothreitol in an NMWL 30 KD ultrafiltration cup at 4°C overnight to a final vol of ca 1 ml (protein concentration, about 1.5 mg/ml). The preparation was stored at -20°C. Isolation of calcium-dependent membrane-binding proteins Five grams of fresh rat brain were homogenized in a Waring blender with 15 ml of 20raM Tris-HC1 buffer (pH 7.5, containing 50mM 2-mercaptoethanol, 1 mM CaC1z and 0.01% leupeptin). Debris was removed by centrifugation at 5000g for 10 rain. The resulting supernatant was centrifuged at 100,000g for 1 hr at 4°C. The pellet was

Inhibition of tyrosine kinase by (+)-aeroplysinin-1 resuspended in 20mM Tris-HCl buffer, pH7.5, supplemented with 10mM EDTA, I mM ATP and 2 m M PMSF (Fava and Cohen, 1984), sonicated briefly (Brown sonifier, at setting 4) and stirred gently for llar. After a further centrifugation step at 100,000g for 1 hr at 4°C, solid (NH4)2SO 4 was added to the supematant until a saturation of 45% (w/v) was reached. The supernatant obtained after centrifugation (35,000g, 10min, 4°C) was concentrated overnight at 4°C using an ultrafiltration tube (NMWL 10 KD; Millipore). Binding o f proteins to erythrocyte inside-out vesicles Human erythrocyte inside-out vesicles were prepared as described (Steck and Kant, 1974). The vesicles were resuspended in 20 mM Tris-HCl buffer, pH 7.5, containing 3raM MgCl2, 2 m M CaC12 and 0.01% leupeptin. Incubation of the vesicles in this buffer with the calciumdependent membrane-binding proteins was performed for l0 min at 22°C. Subsequently the mixture was centrifuged at 40,000g for 15 min at 4°C. The bound proteins were released after resuspension of the pellet in 20 mM Tris-HCl buffer, pH 7.5, containing 10mM EDTA and l mM ATP, and incubated for 15 min at room temperature. The suspension was then centrifuged at 140,000g for 30min at 4°C. The supernatant was concentrated again using an NMWL l0 KD ultrafiltration cup to a final vol of ca l ml (protein concentration, ca 4 mg/ml) and stored at -20°C. Protein kinase assay Assays were performed in a final vol of 500/zl containing 50/zl of solubilized membrane preparation (1.5 mg/ml) and/or 50, 80 or 150/~g/ml of calcium-dependent membrane-binding protein preparation in 20 mM Hepes pH 7.4, 2 mM MgCl2, 1 mM CaCl2, l0/~M sodium vanadate and, if indicated, 0.2 mg/ml of bovine serum albumin. Some assays were supplemented with EGF (20ng/ml) and/or (+)-aeroplysinin-I (0.5/zM). Reactions were started by addition of ATP (15/zM; 5 - 1 2 x 105cpm/assay) and, if indicated, protein substrate and/or EGF. Incubations were performed at 22°C for 10 min. The reactions were terminated by addition of 100/~l of ice-cold 25% trichloroacetic acid. The pellet obtained after centrifugation (10,000g; 4rain) was resuspended in 100/d 1 N NaOH, shaken for 5 min, then treated with 300/tl acetone, and subsequently centrifuged again. The resulting pellets were washed twice in acetone, centrifuged and counted or subjected to gel electrophoresis followed by autoradiography, as described previously (Schr6der et al., 1989, 1990).

153

equilibrated with nitrogen and incubated for 2 hr at 100°C under nitrogen, essentially as described (Hunter and Sefton, 1980). The HCI was removed under reduced pressure and the hydrolysates were dissolved in a marker mixture containing unlabeled phosphoserine, phosphothreonine and 4-phosphotyrosine (Sigma). The phosphorylated amino acids were freed from residual amounts of KCI, HC1, free phosphate and incompletely hydrolyzed proteins by preparative layer chromatography (silica gel 60 plates; Merck) using butanol/methanol/triethylphosphate/acetone/water (10:70: 5 : I0: 5) as solvent. The phosphoamino acid spots visualized by ninhydrin staining were eluted with a mixture of methanol/acetone/water/formic acid (60:25:10:5) at 60°C. After evaporation under reduced pressure labeled phosphoamino acids were analyzed on cellulose thin-layer plates developed with methanol/acetone/H 20/formic acid/acetic acid/butanol (65 : 10:I0 : 5 : 5 : 5). The dried plates were stained with ninhydrin and autoradiographed using Kodak X-Omat film. lmmunoprecipitation Antibodies from anti-calelectrin antiserum (100/~1) were adsorbed to l ml of protein A-Sepharose (10min, 4°C). Subsequently a trichloroacetic acid precipitate, dissolved in 200/~1 PBS, of the protein kinase assay mixture, containing calcium-dependent membrane-binding protein phosphorylated by the solubilized shed membrane-associated tyrosine kinase, was added. After incubation for 20 min on ice and washing with PBS, bound proteins were eluted with 0.5 M KCI in PBS. Proteins were concentrated by trichloroacetic acid precipitation and analyzed by SDS-gel electrophoresis, followed by autoradiography of the dried gels. Analytical methods Electrophoresis of proteins was performed in SDS/10% polyacrylamide gels using Laemmli's procedure (Laemmli, 1970). The separated proteins were transferred to ImmobiIon (Millipore) essentially as described by Towbin et al. (1979). Calcium-dependent proteins were detected in Western blots using polyclonal anti-calelectrin antiserum from rabbits. Biotinylated anti-rabbit IgG, conjugated to streptavidin peroxidase complex (Amersham) was used as a second antibody. Immunocomplexes formed were visualized by addition of 4-chloronaphthol and H202. Protein concentration was determined as described by Lowry et al. (1951), using bovine serum albumin as a standard. RESULTS

Phosphoamino acid analysis The reaction mixtures (final vol 500/~1) consisted of 100/~l calcium-dependent membrane-binding proteins (4 mg/ml) and 15/~M [y32p] ATP (5 - 12 x 105cpm/assay) dissolved in 20mM Hepes pH 7.4, 2 mM MgC12, 1 mM CaCl 2 and l0/~M sodium vanadate. Reactions were started by addition of 100/~l of solubilized membrane preparation and were performed in the absence or presence of EGF (50ng/ml) and/or (+)-aeroplysinin-I (0.5#M) at 22°C for 15min in 1.5-ml mobicol reaction/filtration tubes (Millipore). Subsequently 10 # 1of undiluted anti-calelectrin antiserum was added. After shaking for 15 min at 4°C, the assay mixture was treated with 200/11 of protein A-Sepharose equilibrated with phosphate-buffered saline (20 min on ice). Then the supernatant was pressed out using a syringe, followed by washing several times with 500/~1 PBS each, until no radioactivity was detectable in the eluate. The retained proteins were eluted, under shaking for 5 min and subsequent pressing out, twice with 500 mM KCI and then twice with l M KCI followed by l M HCl. After precipitation with trichloroacetic acid the samples were neutralized with NaOH. The samples were then brought to 5 N HCI, CBP 97/1~-K

Effect on E G F - i n d u c e d cell proliferation The data summarized in Fig. 2 show that in the c o n c e n t r a t i o n range 0.05-0.25/~ M ( + ) - a e r o p l y s i n i n 1 selectively inhibits E G F - c a u s e d D N A synthesis in synchronized M C F - 7 cells. In an earlier report we d e m o n s t r a t e d t h a t ( + ) - a e r o p l y s i n i n - 1 u n d e r otherwise identical conditions at a 10-fold higher concentration did not display any cytostatic activity in n o r m a l h u m a n fibroblasts (Kreuter et al., 1989). In this c o n c e n t r a t i o n range the same rates of [3H]dThd i n c o r p o r a t i o n were f o u n d as those determined for cells grown in m e d i u m supplemented with 5 % newb o r n calf serum w i t h o u t c o m p o u n d . The stimulatory activity o f fetal calf serum representing a well k n o w n pool o f a n u m b e r of growth factors (Bozzola a n d Schimpff, 1985) was suppressed by ( + ) - a e r o p l y s i n i n 1 to a similar extent a n d in the same c o n c e n t r a t i o n range as t h a t observed with a c o m b i n a t i o n o f n e w b o r n calf serum a n d E G F (not shown). However, in the presence o f fetal calf serum the cells

154

MATTHIAS-H. KREUTER et al. Table 2. Effect of (+)-aeroplysinin-1 on DNA synthesis in ZR-75-1 cells Concentration DNA content (/ag/ml) (#g/ml)

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Fig. 2. Effect of (+)-aeroplysinin-1 on EGF-caused proliferation of MCF-7 h u m a n breast carcinoma cells. Cells were synchronized and grown in medium supplemented with 5% newborn calf serum ([]), 10% fetal calf serum (A), or 200 ng/ml of E G F plus 5% newborn calf serum (©), or in the presence o f different concentrations of ( + ) aeroplysinin-1 in medium with 5% newborn calf serum plus 200 ng/ml of E G F (O). The reversibility of the inhibition was tested by incubating the cells under the same conditions in the presence of (+)-aeroplysinin-1 for 36 hr; subsequently the c o m p o u n d was washed out and incubation was continued for 72 hr in medium containing 10% fetal calf serum without (+)-aeroplysinin-1 (&). The incorporation o f [3H]dThd was determined as described in Materials and Methods. Each value represents the mean of 12 experiments; the SD was less than 10%. C, control. d i s p l a y e d still a n at least 1 0 % h i g h e r p r o l i f e r a t i o n index, indicating the existence of some further processes w h i c h a r e n o t i n h i b i t e d b y ( + ) - a e r o p l y s i n i n - I . T o d e t e r m i n e w h e t h e r t h e i n h i b i t o r y effect o f ( + ) a e r o p l y s i n i n - 1 o n M C F - 7 cell g r o w t h is reversible t h e cells were i n c u b a t e d for 3 6 h r in t h e p r e s e n c e o f different concentrations of (+)-aeroplysinin-1. The compound was then washed out and new medium p l u s 1 0 % fetal c a l f s e r u m w a s a d d e d a n d i n c u b a t i o n w a s c o n t i n u e d f o r a n a d d i t i o n a l 72 hr. It w a s f o u n d t h a t t h e g r o w t h i n h i b i t o r y effect o f ( + ) - a e r o p l y s i n i n l w a s c o m p l e t e l y reversible (Fig. 2); n e a r l y t h e s a m e [ 3 H ] d T h d i n c o r p o r a t i o n r a t e s were f o u n d as d e t e r -

0.76 0.63 0.41 0.20 0.22 0.21

mined for untreated cells, grown in the presence of fetal calf serum. In addition it was shown that the viability of the cells could be restored after an incubation of the cells for 36 hr in the presence of this compound at a concentration of 0.05 to 0.40/~M (Table 1). As shown in Table 2, (+)-aeroplysinin-1 inhibited dose-dependently D N A synthesis in ZR-75-1 cells. At a concentration of 0.4/~g/ml, the D N A content was reduced to c a 50%. Figure 3 demonstrates the enhancement of [125I]EGF binding to ZR-75-1 cells after treatment of the cells with (+)-aeroplysinin-1. The amount of bound EGF/assay (containing 2 × 105 cells) was calculated from Scatchard plots by computer analysis. The results were based on D N A content/assay. Addition of different concentrations of (+)-aeroplysinin-I resulted in a decrease of both the amount of bound E G F and D N A content. However, there was a strong increase in the ratio between both values with increasing concentrations of the compound (Fig. 3). Maximal accumulation of [125I]EGF was found at 1.2 #g/ml (3.53 #M) of (+)-aeroplysinin-I. 20-

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Table 1. Reversibility of the inhibitory effect of (+)-aeroplysinin-I on viability of MCF-7 cells Viability of cells after incubation for Concentration 36 br ( + 72 hr) 72 hr (#M) (%) (%) --

100

100

0.05 105 93 0.10 100 87 0.15 103 89 0.20 100 89 0.25 100 68 0.30 86 36 0.35 84 17 0.40 69 3 MCF-7 cells were incubated with different concentrations of (+)aeroplysinin-1 for 36 or 72 hr. After the 36-hr incubation period, the cells were washed and fresh medium without compound, supplemented with 10% fetal calf serum, was added. The incubation was then continued for an additional 72 hr. The viability of the cells was estimated by Trypan Blue exclusion. The controls were set at 100%. The SD was less than 10%.

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Fig. 3. Accumulation of E G F receptor by ( + )-aeroplysinin- I treatment of ZR-75-1 cells. Cells (2 × 105/assay) were incubated in the presence of different concentrations of ( + ) aeroplysinin-I for 2 4 h r and binding of [nSI]EGF (fg//~g of D N A ) was determined as described in Materials and Methods. Mean values from eight quadruplicate determinations are given; the SD was less than 10%.

Inhibition of tyrosine kinase by (+)-aeroplysinin-1

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Fig. 4. Western blot detection of calcium-dependent proteins bound to erythrocyte inside-out vesicles. Calciumdependent proteins were isolated from rat brain by binding to erythrocyte inside-out vesicles as described in Materials and Methods. (a) Coomassie-stained gel of the calciumdependent membrane-binding protein preparation (40/~g). (b) Western analysis of the bound proteins using polyclonal anti-calelectrin antiserum. At higher concentrations, the amount of bound E G F per pg D N A decreased, possibly due to the cytotoxic effect of this compound. These results indicate that (+)-aeroplysinin-I causes an increase of either the EGF-binding affinity of the receptor or the density of the E G F binding sites.

Effect on EGF-induced phosphorylation of calcium-dependent membrane-binding proteins Several protein substrates of EGF-dependent tyrosine protein kinases have been isolated (Pepinsky and Sinclair, 1986; Fava and Cohen, 1984; Glenney and Tack, 1985; Glenney, 1986). A common property of these proteins is their ability to bind to cell

155

membranes in dependence on calcium concentration. In the following we investigated whether the dibrominated tyrosine metabolite, (+)-aeroplysinin-l, can interfere with the phosphorylation of protein substrates of the E G F receptor-associated tyrosine kinase. Calcium-dependent membrane-binding proteins from rat brain were purified by reversible binding to erythrocyte inside-out vesicles• The purified proteins were separated by SDS-gel electrophoresis and detected by Western blot procedure using a polyclonal anti-calelectrin antiserum (Fig. 4). Three major proteins (M r 68,000, 57,000, and 34,000) were found to bind to the inside-out vesicles; two of them (Mr 57,000 and 34,000) were recognized by the anticalelectrin antibodies, representing calpactin I (p57) and calpactin II (p34) (Brugge, 1986; Crompton et al., 1988). As shown in Fig. 5, the solubilized shed membrane vesicle-associated protein kinase activity was markedly stimulated by EGF. It was found that the EGF-induced kinase activity of this preparation was strongly inhibited by (+)-aeroplysinin-1. At a concentration of 0.5/~M and in the presence of 20 ng/mi of EGF, this compound reduced the phosphorylation of the calcium-dependent membrane-binding protein preparation, at a concentration of the protein of 150, 80 and 50 pg/ml, by the solubilized kinase to 33, 25 and 60%, resp. In the absence of EGF, the inhibition of the enzyme by (+)-aeroplysinin- 1 amounted to 43, 49 and 26%, resp. In control experiments it was assessed that the calcium-dependent membrane-binding proteins possess no significant endogenous protein kinase activity (Fig. 5). Assuming a mol. wt of the EGF-dependent tyrosine kinase substrate of 68,000 (Crompton et al., 1988; Moore, 1988), a Km value of 3.47 × 106M was calculated for the enzyme in the absence of inhibitor. In the presence of 0.5/~M (+)-aeroplysinin-1, this value increased to 9.05 × 106 M, resulting in a K i value of 0.91/~M (competitive type of inhibition; data not shown). + (+)-Aerop[ysinin-1

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5. Inhibition of solubilized shed membrane vesicle-associated protein kinase activity by (+)-aeroplysinin-1. The solubilized membrane preparation was treated with (1-3, 7-9) or without 20 ng/ml of EGF (4-6, 10-12). 150~ug/ml (1, 4, 7, and 10), 80,ug/ml (2, 5, 8, and 11) or 50#g/ml (3, 6, 9, and 12) of calcium-binding proteins were used as substrates. Reactions were performed in the absence ( - ) or presence of 0.5/~M (+)-aeroplysinin-1. In control experiments, solubilized membrane preparation was substituted by buffer and incubation was performed with (I 3) or without 20 ng/ml of EGF (14). Each value represents the mean of three independent experiments; the SD was less than 10%.

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156

MATTH1AS-H. KREUTER et al.

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P-Thr

68 /,3 35 14.3

Fig. 6. Phosphoprotein pattern of calcium-dependent membrane-binding protein preparation phosphorylated by solubilized shed membrane vesicle-associated protein kinase. Phosphorylation was performed without (lanes Ba and Bc) or with 20 ng/ml of EGF (lanes Bb and Bd) in the absence (lanes Ba and Bb) or presence of 0.5/t M ( + )-aeroplysinin- 1 (lanes Bc and Bd). Samples were subjected either directly (lanes Ba-Bd) or after immunoprecipitation of lipocortinlike proteins by anti-calelectrin antibodies (lanes Ca and Cb; experiments in the presence of 20 ng/ml of EGF only) to electrophoresis on SDS/10% polyacrylamide gels, followed by autoradiography. Shown are the autoradiograms. Lane A, Coomassie-stained gel of Ba-Bd. Mol. wt standards were: myosin (Mr 200,000), fl-galactosidase (130,000), bovine serum albumin (68,000), ovalbumin (43,000), alcohol dehydrogenase (35,000) and lysozyme (14,300). The phosphoprotein pattern of the calciumdependent membrane-binding protein preparation, obtained after phosphorylation with the solubilized shed membrane vesicle-associated protein kinase is shown in Fig. 6. The phosphorylation of these proteins was strongly increased in the presence of E G F (lanes Ba and Bb). Addition of (+)-aeroplysinin-1 (0.5 pM) selectively suppressed the incorporation of phosphate into these proteins. The phosphorylated lipocortin-like proteins contained in the calcium-dependent membrane-binding protein preparation could be immunoprecipitated with the polyclonal anti-calelectrin antibodies (lane Ca). No incorporation of phosphate was detected, if the lipocortin-like proteins were precipitated with the antibodies after phosphorylation in the presence of (+)-aeroplysinin-1 (lane Cb). Determ&ation o f the amino acid substrate

In order to establish that the enzyme activity inhibited by (+)-aeroplysinin-1 indeed represents a tyrosine-specific protein kinase, the calciumdependent proteins phosphorylated by the solubilized membrane preparation were immunoprecipitated by anti-calelectrin antiserum and subjected to phosphoamino acid analysis. As demonstrated in Fig. 7, lane b, only tyrosine is phosphorylated in the lipocortinlike proteins, recognized by anti-calelectrin antibodies, using solubilized protein kinase from shed membrane vesicle preparation. Addition of 0.5 # M (+)-aeroplysinin-1 almost totally suppressed the EGF-dependent phosphorylation of tyrosine residues

P-Ser

Fig. 7. Phosphoamino acid analysis of calcium-dependent proteins phosphorylated by shed membrane vesicle-associated protein kinase. The phosphorylation of calcium-dependent membrane-binding proteins (150/~g/ml; presence of 20 ng/ml of EGF) was performed in the absence (lanes a and b) and in the presence of 0.5/~M (+)-aeroplysinin-1 (lanes c and d). In lanes b and d, the phosphorylated proteins were immunoprecipitated by polyclonal anti-calelectrin antiserum, eluted and then subjected to hydrolysis under acidic conditions. The phosphoamino acids were separated by cellulose thin-layer chromatography as described in Materials and Methods. Shown are autoradiograms of the dried cellulose plates. The positions of the unlabeled phosphoamino acid standards (P-Ser, P-Thr, and P-Tyr) were detected by ninhydrin staining and are indicated at the right. (lanes c and d), while the incorporation of phosphate into threonine residues of total calcium-dependent membrane-binding protein preparation was not affected (cf. lanes a and c). DISCUSSION

There are several reports demonstrating an enhancement of E G F receptor phosphorylation in tumor cells and an involvement of the intrinsic tyrosine-specific kinase activity in this process (Hunter and Sefton, 1980; Earp et al., 1988). Lipocortin-like proteins and some related proteins were shown to be major substrates for tyrosinekinases (Pepinsky and Sinclair, 1986; Fava and Cohen, 1984; Glenney and Tack, 1985; Sawyer and Cohen, 1985; Braun et al., 1984; Veale et al., 1989). The aim of the presented work was to study the influence of (+)-aeroplysinin- 1 on processing of E G F receptor-associated tyrosine-kinase, its substrate phosphorylation and its signal transduction-related stimulation of cell proliferation. Previously we demonstrated that (+)-aeroplysinin-1 displays a potent cytotoxic activity against tumor cells in vitro and a high antitumor activity in the L5178y mouse ascites tumor model in vivo. These effects were found to be selective over a wide concentration range, in which (+)-aeroplysinin-1 displays only cytostatic activity on non-transformed cells (Kreuter et al., 1989). Now we determined that during treatment with (+)-aeroplysinin-1, MCF-7 cells lost their ability for EGF-mediated cell response. Cell death occurred within 36-72 hr. These effects were found to be reversible after removal of the compound. Previously we determined that the rate of incorporation of [3H]dThd of murine spleen lymphocytes and their ability to respond to mitogens (Concanavalin A

Inhibition of tyrosine kinase by (+)-aeroplysinin-I for T-lymphocytes, and lipopolysaccharide for Blymphocytes) are not influenced at an up to 20-fold higher dose than that which effectively inhibits cell response and strongly suppresses [3H]dThd incorporation rate of MCF-7 cells (Kreuter et al., 1989). From these observations we got the idea that aeroplysinin might affect growth factor receptor-associated enzymes and, in consequence, gene expression in transformed cells. One parameter of activation of EGF receptor-associated tyrosine-specific kinase is the ligand-induced endocytosis of the receptor. We found a dramatic increase of EGF binding to aeroplysinin-treated cells compared to the controls, despite a suppressed DNA synthesis, indicating an accumulation of the EGF receptor. Because a substrate phosphorylation is required for this process, initiating subsequent autophosphorylation and Ckinase phosphorylation of the EGF receptor (Glenney et al., 1988; Cochet et aL, 1984), we conclude that (+)-aeroplysinin-1 inhibits some step in this processing. Determinations of protein kinase activity of a solubilized membrane preparation of MCF-7 cells using lipocortin-like proteins as substrates revealed a nearly complete inhibition of tyrosine-specific phosphorylation in the presence of concentrations of (+)-aeroplysinin-I as low as 0.5 pM. The activity of the solubilized kinase was found to respond to treatment with EGF. Applying an immunoprecipitation procedure, lipocortin-like proteins, which are inhibitors of phospholipase A2 activity (Brugge, 1986; Gramzow et al., 1989), were detected as the main phosphorylated substrates. The phosphorylation of these proteins was markedly enhanced after treatment with EGF; it was abolished in the presence of 0.5 # M (+)-aeroplysinin-l. Phosphoamino acid analysis revealed phosphotyrosine to be the only phosphorylated amino acid in immunoprecipitates of lipocortin-like proteins, obtained by using an anti-calelectrin antiserum which also recognized calpactin I and calpactin II. From its ability to inhibit the ligand-dependent intrinsic protein tyrosine kinase activity of EGF receptor, the sponge tyrosine metabolite (+)-aeroplysinin-1 appears to be a promising compound in a rational chemotherapy. Further studies must show whether (+)-aeroplysinin-1 also inhibits other protein tyrosine kinase activities found to be present in many oncogene products. Acknowledgements--This work was supported by a grant

from the Bundesministerium ffir Forschung und Technologie ( # 0319207A8, under the co-ordination of the Kernforschungsanlage J/ilich and of the Internationales Bfiro GKSS, Geesthacht). REFERENCES

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