Tamoxifen modulates protein kinase C via oxidative stress in estrogen receptor-negative breast cancer cells.

Nonsteroidal agent tamoxifen (Tam), a therapeutic/chemopreventive agent for breast cancer, inhibits protein kinase C (PKC), which is considered to be one of its extra-estrogen receptor sites of action. This drug is required at higher (>100 μM) concentrations to inhibit PKC in the test tube, whereas it is required at lower (1-10 μM) concentrations to induce inhibition of cell growth in estrogen receptor-negative cell types. To identify additional mechanisms of action of Tam on PKC and cell growth, studies with MDA-MB-231, an estrogen receptor-negative breast carcinoma cell type, have been carried out. Upon treatment with 5-20 μM Tam, a cytosol to membrane translocation of PKC occurred within 30 min, which was then followed by a down-regulation of the enzyme within 2 h. A transient generation of Ca2+/lipid-independent activated form of PKC was observed during this period. Rapidly growing cells require nearly 2-3-fold lower concentrations (2-5 μM) of Tam than do confluent cells to induce changes in PKC. Furthermore, phorbol ester binding observed with intact cells also decreased in Tam-treated cells only under the conditions PKC was inactivated. Unlike phorbol esters, Tam did not directly support the membrane association of PKC. The release of arachidonic acid correlated with the PKC membrane translocation. Studies carried out with [3H]Tam revealed that Tam partitioned into the membrane, and there was no appreciable covalent association of [3H]Tam with cellular proteins within this limited time period (2 h). Various antioxidants (vitamin E, vitamin C, β-carotene, catalase, and superoxide dismutase) inhibited all these cellular effects of Tam. Moreover, vitamin E strikingly blocked Tam-induced growth inhibition. To determine whether oxymetabolites of Tam can affect PKC permanently, OH-Tam was tested with purified PKC. In contrast to Tam, which reversibly inhibited PKC, OH-Tam permanently inactivated the enzyme by modifying the catalytic domain at lower concentrations. The vicinal thiols present within this domain were found to be required to induce this inactivation. This effect was partially blocked by various antioxidants. This is the first report showing the role of oxidative stress in mediating the actions of Tam. Taken together these results suggest that Tam, by initially partitioning into the membranes, induces a generation of transmembrane signals and an oxidative stress to elicit the membrane association of PKC, followed by an irreversible activation, and subsequent down-regulation of this enzyme, which, in part, may lead to cell growth inhibition.

tially partitioning into the membranes, induces a generation of transmembrane signals and an oxidative stress to elicit the membrane association of PKC, followed by an irreversible activation, and subsequent down-regulation of this enzyme, which, in part, may lead to cell growth inhibition.
Tamoxifen (Tam) 1 is a synthetic nonsteroidal anti-estrogen that is widely used for the chemotherapy of breast cancer and is currently being evaluated for chemoprevention of this disease (1)(2)(3). The antiproliferative actions of Tam and other triphenylethylene derivatives at submicromolar concentrations in estrogen-dependent breast carcinoma cells are believed to be mediated by high affinity binding to the estrogen receptor (ER) (4 -8). The effects induced by submicromolar concentrations of Tam can be overcome by a high concentration of estrogen (4 -8). However, Tam also inhibits growth of ER-negative cell lines at low (1-10 M) concentrations, which is not overcome by estrogen (8 -11). Furthermore, it inhibits growth of other cell types, which have no ER, in some cases at nanomolar concentrations (11). The mechanism of such ER-independent inhibition of tumor cell growth by Tam is not clearly known. Binding to so-called anti-estrogen sites and the inhibitions of calmodulin and protein kinase C (PKC) are considered to be some known additional sites of action of Tam-related agents (12)(13)(14)(15)(16).
Since PKC may play a crucial role in the signal transduction that influences cell growth and transformation (17,18), the observation of the inhibition of PKC by Tam has gained considerable attention. Purified PKC has been shown to be reversibly inhibited by Tam-related agents in the test tube (15,16). This inhibition seems to involve a number of different mechanisms including binding of the drug to phospholipids that are required for activation, binding to the catalytic domain at ATPbinding site, and binding to the regulatory domain (15, 16, 19 -21). Moreover, the modes of action have been found to be slightly different for individual types of triphenylethylene antiestrogens tested (22). The metabolites of Tam, 4-hydroxytamoxifen (OH-Tam) and N-demethyltamoxifen, have also been shown to inhibit PKC in a reversible manner (23). The inhibition of PKC in the test tube requires higher concentrations of Tam (IC 50 100 M) (15), while the ER-independent cell growth inhibition requires only 1-5 M Tam. This raises the possibility that Tam induces growth inhibition at this low concentrations by acting on cellular targets other than PKC and/or by inducing other cellular mechanism(s), which may complement its action at PKC.
We have been involved in studies of regulation of PKC by oxygen radicals and thiol modifying agents (24,25). Such studies also facilitated understanding of the mechanisms of inactivation of PKC by its commonly used inhibitors, calphostin C, hypericin, and chelerythrine. These inhibitors induce an irreversible inactivation of PKC either involving oxygen radical production or alkylating mechanisms (24). PKC inhibitors are classified based on their site of action, either related to regulatory or to catalytic domains. Based on the mechanism of action, these inhibitors also can be classified as reversible inhibitors or irreversible inactivators (24). However, in order to identify irreversible inhibitors, it is not only necessary to evaluate them using an isolated enzyme in the test tube, it is also important to study them in intact cells. A reversible inhibitor in test tube could become an irreversible inactivator after metabolic activation. This is especially important for Tam, which is known to be oxidatively metabolized in the cell and can form reactive metabolites that can covalently bind to DNA and proteins (26 -29).
Tamoxifen also has been shown to stimulate the Ca 2ϩ /phospholipid-dependent PKC activity in test tube at high concentrations of Ca 2ϩ (30). Unlike in test tubes, where Tam inhibited PKC-mediated phosphorylation of proteins, with intact cells Tam did not inhibit phorbol ester-induced phosphorylation of endogenous proteins, but instead by itself stimulated the phosphorylation of some endogenous proteins (31). Recent studies have shown that staurosporine, an inhibitor for PKC in a test tube, could function as an activator of PKC in intact cells inducing the membrane translocation of PKC (32)(33)(34). Staurosporine also induced a release of arachidonic acid, which is a common effect induced by a variety of structurally unrelated tumor promoters (32,35). In fact, in the mouse skin carcinogenesis model, staurosporine acts as a tumor promoter (32,33). In this contest it is noteworthy that Tam acts as a chemopreventive agent in mammary carcinogenesis, while it can also induce uterine cancer in humans as well as liver cancer in rats (10, 36 -38). Therefore, it is important to extend the studies of Tam to intact cells to understand whether it directly or after metabolic activation can influence PKC or the upstream signal transduction mechanisms in a bimodel manner. However, to date, such studies have not been carried out with Tam in intact cells.
In this report we show that Tam and other related agents at low (1-10 M) concentrations can induce the membrane association of PKC, the irreversible activation and the subsequent down-regulation of the enzyme involving the generation of transmembrane signals, and oxidative regulation in a ER-negative breast cancer cell line, MBA-MB-231. Unlike Tam, which inhibits PKC in a reversible way, at least one of its oxymetabolites, 4-hydroxytamoxifen (OH-Tam), can induce an irreversible oxidative inactivation of the isolated kinase, probably by interacting with the vicinal thiols present within the catalytic domain.

EXPERIMENTAL PROCEDURES
Materials-Tamoxifen (trans) citrate, catalase from bovine liver, and superoxide dismutase from bovine liver were obtained from Sigma. Vitamin E (D-␣-tocopherol) and ␤-carotene were from Fluka. [N-methyl- 3 H]Tamoxifen citrate (specific activity 84 Ci/mmol) was from Amersham Corp.; [20-3 H]phorbol 12,13-dibutyrate (specific activity 20 Ci/ mmol), and myo- [2-3 H]inositol (specific activity 17.5 Ci/mmol) [5,6,8,9,11,12,  Rabbit brain PKC (a mixture of ␣, ␤, and ␥ isoenzymes) was purified as described previously (24). Unless otherwise mentioned, unfractionated mixture of these Ca 2ϩ -dependent isoenzymes of PKC was used for modification studies. In some cases individual isoenzymes separated by hydroxylapatite (39) were used. The catalytic and regulatory domains were separated after treating PKC with a low (1 g/ml) concentration of trypsin (19). DEAE-Cellulose Chromatographic Isolation of PKC from Cells Treated with Tam-Cells were grown in 100-mm Petri dishes in minimal essential medium with Earl's salts (MEM) supplemented with 5% fetal calf serum. Either subconfluent (25%) or confluent cells were changed to fresh serum-free MEM and incubated with various concentrations of Tam, OH-Tam, or control vehicle (ethanol) for the indicated periods of time at 37°C. Then the soluble and detergent-solubilized membrane fractions were prepared from the treated cells and subjected to DEAE-cellulose chromatography as described previously (40). The Ca 2ϩ /phospholipid-stimulated PKC activity (proform) was eluted using 0.1 M NaCl (peak A) and the Ca 2ϩ /phospholipid-independent and the modified PKC activity was eluted with 0.25 M NaCl (peak B).
PKC Assay-The assays of PKC as well as cAMP-dependent protein kinase were carried out in 96-well plates with fitted filtration discs made of Durapore membranes (40). Briefly, PKC reaction samples containing 20 mM Tris-HCl, pH 7.5, 10 mM MgCl 2 , 0.33 mM CaCl 2 , 0.1 mM ATP, histone H1 (0.1 mg/ml), 0.04 M leupeptin, and 25 l of PKC sample in a total volume of 125 l were incubated at 30°C for 5 min. PKC activity was expressed as units, where 1 unit of enzyme transfers 1 nmol of phosphate to histone H1/min at 30°C. Since protein phosphatase 2A activity was high in the peak B fraction, microcystin-LR (100 nM) was included during PKC assay to obtain a reliable measure of histone H1 phosphotransferase activity in this fraction.
Phorbol Ester Binding with Isolated PKC-[ 3 H]Phorbol 12,13-dibutyrate (PDBu) was used as a ligand for the determination of phorbol ester binding, using the multiwell filtration approach (40). For optimal PDBu binding to purified PKC, the conditions discussed in method 1, described previously, were used (40). To determine the optimal PDBu binding to PKC fraction that was isolated by DEAE-cellulose chromatography from the crude cell extracts, the conditions standardized with the cytosolic receptor (method 2) were used (40).
Phorbol Ester Binding in Intact Cells-Cells were grown in 35-mm Petri dishes. Sets of three Petri dishes were used for determining specific and nonspecific bindings. The medium was changed to a fresh MEM (no serum), and then the cells were treated with various concentrations of Tam for a 2-h time period. Then 37.5 nM [ 3 H]PDBu (0.25 Ci) was added to the medium and cells were further incubated for 45 min. For determining nonspecific binding 10 M unlabeled PDBu was included with radiolabel. After incubations, cells were washed four times with ice-cold saline and lysed with 0.2 M NaOH, and the radioactivity present in the cell extract was determined. The specific binding was calculated by subtracting the nonspecific binding from the observed total binding. PKC Membrane Binding with Homogenates and Isolated Membrane-Whether Tam or OH-Tam induce chelator-stable membrane association of PKC in the homogenates or with isolated membrane using purified PKC was determined using previously described procedures (41). Briefly, crude cell homogenates were incubated with Tam or OH-Tam (10 M) or PMA (100 nM) in the presence of 0.1 mM CaCl 2 at room temperature for 10 min. Then EGTA was added to the homogenate, and the membrane and cytosolic fractions were separated to determine PKC activity associated with these fractions. Similarly, experiments were conducted using the isolated membrane and purified PKC.
Covalent Binding of Tam to Proteins in Intact Cells-Subconfluent cells (10 -15 ϫ 10 6 ) were incubated with 25 M [ 3 H]Tam (3.75 Ci) for a 2-h period at 37°C in a serum-free MEM to allow substantial inactivation of PKC. Then, under these conditions, whether Tam covalently bound to protein or not was determined. The cells treated with [ 3 H]Tam were washed four times with saline and then scrapped into 10 ml of saline and collected by centrifugation at 2,000 ϫ g for 5 min. The cell pellet was then lysed with 3 ml of cold acetone by sonication, and then the protein was allowed to precipitate by keeping it at Ϫ10°C for 30 min. The precipitated protein was collected by centrifugation. The protein pellet was washed twice with each organic solvent, acetone, nhexane, and methanol. The washed protein pellet was suspended in SDS-polyacrylamide gel electrophoresis sample buffer without mercaptocompounds and heated at 100°C for 3 min to solubilize the protein, and then the radioactivity was counted. To determine a covalent binding of [ 3 H]Tam to PKC, the detergent-solubilized cell extract was prepared from the cells treated with [ 3 H]Tam and it was subjected to the centrifuge column technique to remove free Tam. The protein fraction was incubated with rabbit polyclonal antibodies raised against PKC (mixture of ␣, ␤, and ␥) at 30°C for 4 h, and then the antibody-PKC complex was isolated by incubation with protein A-Sepharose, followed by centrifugation. From the Sepharose beads, protein was extracted with SDS-polyacrylamide gel electrophoresis sample buffer and the radioactivity was counted.
Modification of Purified PKC with OH-Tam-Initially mercaptocompounds present within the PKC preparation were removed by using a PD-10 gel filtration column (Pharmacia Biotech Inc.). The column chromatography also facilitated the exchange of buffer in PKC preparation to buffer consisting of 20 mM Tris-HCl, pH 7.5, 0.1 mM EGTA, 150 nM pepstatin, 1 M leupeptin. PKC (approximately 1-1.5 units) was incubated with various concentrations of Tam or OH-Tam in the presence of 1 mM CaCl 2 in a total volume of 0.5 ml for 10 min at 37°C. Then 25 l of bovine serum albumin solution (10 mg/ml) and 25 l of 0.1 M EDTA in 50 mM Tris-HCl, pH 8, were added. 2 Tam and other low molecular weight compounds present in the treated PKC samples were removed by subjecting them to the centrifuge column technique using Sephadex G-50 (24). The used Sephadex G-50 columns were washed with 2 bed volumes of ethanol to remove the trapped Tam in the gel and then equilibrated with buffer. This ethanol wash to remove Tam that retained within the gel was required to reuse the column, which otherwise would result in a low recovery of PKC from the column upon subsequent reuse.
Arachidonic Acid Release-Cells were grown to subconfluence or confluence in a regular medium in 96-well plates. The medium was then replaced with MEM with 0.1% serum and [ 3 H] arachidonic acid, 0.5 Ci/ml medium, and the cells were incubated for 4 h at 37°C. Then the cells were washed four times with MEM medium. The labeled cells (four wells for each point) were incubated with MEM supplemented with 0.1% serum along with various concentrations of Tam. At the indicated periods of time, the medium was transferred from the wells of the culture plate to appropriate wells in a 96-well filtration plate fitted with Durapore membrane filtration disks. Then the samples were filtered into an ordinary 96-well plate by using a minivacuum manifold (Millipore). The filters removed any radiolabeled cells that were detached during the incubation of the cells. The radioactivity present in the filtrates was then counted. 45 Ca Uptake-Initially, MDA-MB-231 cells were grown to confluence in 96-well plates. The serum-containing medium was removed. Then serum-free MEM containing 45 CaCl 2 (2.5 Ci/ml) along with various concentrations of Tam was added to the wells, and the cells were incubated at 37°C. At various time intervals, the medium was quickly removed and then the cells were washed three times with 150 l of MEM containing 1 mM LaCl 3 . Then the cells were lysed with 0.2 M NaOH, and the radioactivity retained within the cell was counted.
Inositol Phospholipid Hydrolysis-Cells were grown in 35-mm Petri dishes and labeled overnight with myo-[2-3 H]inositol (10 Ci/ml) in a inositol-free MEM supplemented with dialyzed FCS (5%). Cells were washed and treated with various concentrations of Tam for given periods of time in a serum-free MEM supplemented with 10 mM LiCl. The treated cells were then washed and extracted with perchloric acid. From the neutralized perchloric acid extracts, inositol phosphates were separated by ion-exchange chromatography using Dowex-1 (42).
Growth Inhibition Assays-The size of the intracellular thymidine pool was shown to be reduced by Tam, which can cause artifacts in thymidine incorporation into the cell (43). Therefore, two approaches were used to measure the cell growth rate. Cells were seeded in 96-well multiwell plate to 25% confluence in MEM supplemented with 5% FCS. After allowing the cells to attach, various concentrations of Tam were added (four wells for each point) and the cells were incubated for 48 h at 37°C. Then, from one set of multiwell plates, the medium was removed and the cells were incubated with MEM supplemented with 5% dialyzed FCS and 0.5 Ci/ml [methyl-3 H]thymidine. After 6 h of incubation, the cells were washed four times with saline and lysed with 0.2 M NaOH and the radioactivity retained in the cells was counted. Alternatively, cells treated with Tam in another set of multiwell plates were stained with sulforhodamine B, and the absorbance was measured at 550 nm using Thermomax multiwell plate reader as a growth index (44).

Membrane Association and Down-regulation of PKC Induced by Tam and OH-Tam-An
ER-negative cell line, MDA-MB-231, was employed to understand ER-independent effects of Tam-related agents on PKC. The changes that occurred with the treatment of Tam and OH-Tam were compared with that of tumor promoter, phorbol 12-myristate 13-acetate (PMA). In confluent cells treated in a serum-free medium with 20 M Tam or OH-Tam, there was a substantial decrease in the activity of PKC in the cytosol fraction within 30 min (Fig. 1). A concomitant increase in the activity of PKC in the membrane fraction was found. With Tam treatment, the extent of membrane translocation of PKC was not appreciable up to 15 min. However, PMA-induced membrane association of PKC occurred within 15 min. In another experiment carried out with treatments for limited time (2 and 5 min), only PMA induced PKC translocation within these short periods of time but not Tam and OH-Tam. Thus, there was a lag period of nearly 15 min in inducing the membrane translocation of PKC by Tam. A prolonged treatment by both Tam and OH-Tam induced a pronounced decrease in PKC activity in both cytosol and membrane. In the PMA-treated cells, however, there was no decrease in total (cytosol ϩ membrane) PKC activity during this 2-h time period. Nevertheless, by extending the PMA treatment to 6 h, there was a substantial (85%) decrease in the total PKC activity. The extent of membrane translocation of PKC induced by OH-Tam was slightly lower than that observed with Tam. This might be due to a rapid onset of down-regula- tion of PKC induced by OH-Tam. Moreover, in all these cases PKC-associated [ 3 H]PDBu binding present within the peak A fraction eluted from DEAE-cellulose showed a similar pattern of changes in the cytosol and membrane (data not shown), suggesting that a true physical redistribution of PKC and inactivation might have occurred. There was no change in the activity of cAMP-dependent protein kinase isolated from these Tam-treated cells.
Generation of a Ca 2ϩ /Lipid-independent Activated Form of PKC-Because PMA as well as various oxidants (hydrogen peroxide, m-periodate, and polyphenolic agents) induce a transient formation of a Ca 2ϩ /phospholipid-independent activated form of PKC prior to an induction of appreciable down-regulation of the enzyme (24, 25), we have determined whether such an activated form was also formed during the treatment with Tam. In confluent MDA-MB-231 cells treated with Tam (20 M), there was a decrease in Ca 2ϩ /phospholipid-dependent PKC activity that eluted with 0.1 M NaCl (peak A) within 1 h of Tam treatment (Fig. 2). Concomitantly, the modified form exhibiting a lesser dependence on Ca 2ϩ /phospholipid (peak B) increased. This peak B activity eventually declined at a later time period (2 h). The Ca 2ϩ /phospholipid-independent histone phosphotransferase activity that transiently elevated in the peak B was also inhibited by the pseudosubstrate peptide (PKC residues 19 -31).
Cell Growth Rate and Susceptibility to Tam-induced Changes in PKC-Given the fact that cell growth inhibition occurs at concentrations (5-10 M) lower than that of the concentrations required to inactivate PKC in confluent cells within a 2-h period, it is possible that PKC inactivation by Tam may be more sensitive in rapidly growing subconfluent cells than that in confluent cells. Since the membrane-associated PKC activity was high in subconfluent cells, the extent Tam-induced cytosolto-membrane translocation of PKC was less. However, subconfluent cells were 2-3-fold more sensitive to down-regulation by Tam than were confluent cells (data not shown). For example, in order to induce a 50% down-regulation of PKC, Tam was required at 15 M with confluent cells, while it required only 5 M with subconfluent cells. Since membrane association of PKC was high in the rapidly growing cells, it is possible that prior membrane association of PKC might have enhanced subsequent down-regulation of PKC at a lower concentration of Tam. Alternatively, it is possible that the metabolic activation of Tam may be higher in the rapidly growing cells to accelerate the process of inactivation of PKC.
PMA-induced Membrane Association of PKC Promoted Its Inactivation by Tam-To determine whether prior membrane association of PKC enhances the rate of the down-regulation of PKC by Tam, PMA was used to induce an initial membrane translocation of PKC. Since PMA by itself can induce the downregulation of PKC with a prolonged treatment, the pretreatment with PMA was restricted to a limited time just enough to induce only the membrane association of PKC but not its downregulation. When confluent cells were treated with Tam (20 M) alone, there was only a 22% decrease in the total (cytosol ϩ membrane) PKC activity within a 1-h time period (Fig. 3). However, Tam, when coadministered with PMA, inactivated PKC by 64% within this time. This suggested that PMA can enhance Tam-induced inactivation of PKC by facilitating the initial membrane association of PKC.
Indirect Action of Tam on PKC Translocation-Previous studies carried out by this laboratory have shown that PMA could induce a chelator-stable membrane association of PKC by directly binding to PKC-lipid complex (41). To determine whether Tam or other related agents could induce chelatorstable membrane association of PKC in a manner similar to phorbol esters, experiments were carried out using crude cell homogenates and isolated membrane incubated with purified PKC. Only PMA in the presence of Ca 2ϩ produced the chelatorstable membrane association of PKC, while Tam and other related agents failed to promote the membrane binding of PKC either in the crude homogenates or with the isolated membranes.
Decrease in PDBu Binding in Intact Cells in Relation to Irreversible Inactivation of PKC-The phorbol ester binding determined with intact cells represents the state of PKC within the cells. Therefore, we have determined [ 3 H]PDBu binding in cells treated with Tam. In cells treated with Tam, there was a decrease in PDBu binding correlating with inactivation of PKC under these conditions (Fig. 4). In a previous study, a decrease in [ 3 H]PDBu binding in intact cells was observed with Tam concentrations that were far lower than that required to inhibit purified PKC (15). It was assumed that the inhibition of PDBu binding occurred directly by Tam in a manner similar to PKC inhibition that occurred in the test tube. However, with isolated PKC, the PDBu binding was not affected by Tam (19). The current study suggested that the observed inhibition of PDBu binding in other cell types tested reflects an irreversible inactivation of PKC occurring at a lower concentration of Tam in the cells rather than due to a direct interference of PDBu binding by Tam.
Second Messengers That Are Involved in Tam-induced Membrane Association of PKC-Since Tam cannot promote a direct association of PKC with the isolated membranes, and since the observed membrane association of PKC in the Tam-treated cells occurred with a lag of nearly 30 min, a generation of endogenous agents in response to Tam treatment might have facilitated the membrane association of PKC. Given the fact that second messengers such as Ca 2ϩ , diacylglycerol, and arachidonic acid support membrane association of PKC, we have determined 45 Ca 2ϩ uptake, hydrolysis of phosphatidylinositol, and arachidonic acid release in Tam-treated cells. In MDA-MB-231 cells treated with Tam, there was no increase in 45 Ca 2ϩ uptake compared to control cells. Similarly, the hydrol-ysis of inositol phospholipids was not altered as measured by the radioactive label in inositol phosphates. However, in cells treated with Tam, there was a substantial increase in the release of [ 3 H]arachidonic acid from the cells prelabeled with [ 3 H]arachidonic acid (Fig. 5).

Covalent Binding of [ 3 H]Tam to PKC-Subconfluent cells were incubated with 25 M [ 3 H]Tam (3.75
Ci) for various periods of time (5-120 min) at 37°C. The cytosolic and membrane fractions were initially isolated from the cells treated with [ 3 H]Tam by centrifugation, and then acetone was used to extract the membrane associated Tam. Approximately 6 -9 nmol/10 6 cells (85-95% of the total radioactivity) was recovered with acetone from the membrane fraction, while 0.2-1.1 nmol/ 10 6 cells (5-15% of the radioactivity) was recovered in the cytosolic fraction (data not shown). This suggested that Tam may initially partition into the membrane and then mediate its effects leading to initial membrane association of PKC. The radioactivity associated with the acetone-precipitated protein fraction was low (0.05% of the total radioactivity that was retained in the cell). Furthermore, there was no detectable amount of the radioactivity in the immunoprecipitated PKC fraction from the crude cell homogenate. Since the cell labeling with [ 3 H]Tam was carried out for only 2 h, we cannot exclude the possibility of increased covalent binding of [ 3 H]Tam with PKC or other macromolecules with prolonged incubation period. However, the covalent binding to PKC may not necessarily be the sole mechanism for inactivation of PKC observed in the cells treated with Tam for limited time (2 h).
Protection of Tam-induced Inactivation of PKC by Antioxidants-Since Tam is known to go through oxidative metabolism, it is possible that some oxymetabolites may produce reactive oxygen species and affect PKC in an irreversible manner. Formation of 4-monohydroxy-, 3,4-dihydroxy-, and Noxide metabolites have been previously reported (1). Therefore, we tested both water-soluble and lipid-soluble antioxidants to block the Tam-mediated inactivation of PKC in MDA-MB-231 breast carcinoma cells. Since the translocation was a transient process, to better evaluate the antioxidant protective effects, the effect of these agents on the down-regulation of total (cytosol ϩ membrane) PKC activity was determined initially. N-Acetylcysteine had no effect on the rate of inactivation of PKC  induced by Tam, whereas vitamin C, vitamin E (D-␣-tocopherol), and ␤-carotene all inhibited the down-regulation of PKC (Fig. 6). Furthermore, antioxidant enzymes, SOD, and catalase were partially effective in preventing the Tam-induced inactivation of PKC in both cell types. Conceivably, an oxidative process may be involved in the irreversible inactivation of PKC occurring in the Tam-treated cells. Inhibition of this process by different antioxidant systems suggests the formation of different reactive oxygen species in the Tam-treated cells.
Whether antioxidants inhibited PKC translocation prior to blocking the PKC down-regulation was studied using vitamin E, which produced the best protective affect. Vitamin E decreased the extent of the translocation of PKC occurring within a 30-min time period (data not shown). Therefore, it is possible that vitamin E, by decreasing the oxidative stress, prevented membrane association of PKC. Furthermore, vitamin E also decreased arachidonic acid release from the cells (Fig. 7). There is a possibility that inhibition of Tam-induced oxidative stress may prevent the subsequent activation of phospholipases A 2 and D, leading to a decrease in the release of arachidonic acid from the membrane phospholipids. However, we cannot exclude the possibility that vitamin E directly inhibited phospholipase A2 activity as has been shown by others (45).
Since vitamin E inhibits several effects of Tam, whether vitamin E inhibits the intake and retention of Tam in the cell was determined. Even at higher concentrations (100 M) of vitamin E inhibited only 5-10% [ 3 H]Tam retention within the cell (data not shown). Such a small effect of vitamin E on Tam retention is unlikely to be responsible for its dramatic effects on PKC and cell growth.
Vitamin E Blocks Tam-induced Cell Growth Inhibition-Since vitamin E blocks the Tam-mediated oxidative regulation of PKC, we have determined whether vitamin E can block the Tam-induced growth inhibition of the cells. Vitamin E alone had no growth inhibitory effect on the cell growth. However, when coincubated with Tam, vitamin E blocked nearly 80 -90% of the Tam-induced cell growth inhibition (Fig. 8). This strongly suggests that Tam-mediated growth inhibition might have been mediated by oxidative stress occurring in the membrane.
Irreversible Inactivation of Purified PKC by 4-Hydroxytamoxifen-Since Tam-induced inhibition of PKC activity in test tube is totally reversible, the observed irreversible inactivation of PKC occurred in intact cells treated with Tam was unlikely to be caused by a direct action of unmodified drug. It is possible that either the endogenous agents that are elevated in response to Tam treatment or the metabolites of Tam might have induced this irreversible inactivation of PKC. Among the oxymetabolites of Tam, OH-Tam was extensively studied (1). Therefore, we determined whether or not OH-Tam can induce an irreversible inactivation of purified PKC in test tube. Rabbit brain PKC (a mixture of ␣, ␤, and ␥ isoenzymes) was incubated with either Tam or OH-Tam under defined conditions, and after removing the drug from the treated PKC preparation, both kinase activity and PDBu binding were determined. Thus, at a later step during the determination of kinase activity and PDBu binding, the drug was no longer present. When incubations were carried out in the presence of 0.1 mM EGTA, there was no decrease in either kinase activity or PDBu binding. However, when 1 mM CaCl 2 was included in this incubation, OH-Tam inactivated PKC activity with IC 50 value of approximately 50 M (Fig. 9). The loss of PDBu binding occurred only at higher concentrations with IC 50 of 90 M. Tam did not induce this inactivation of the enzyme, even at a higher (200 M) concentration. PKC activity did not recover by subjecting the OH-Tam treated PKC to extensive dialysis, to DEAE-cellulose chromatography in the presence of Nonidet P-40, or to hydrophobic interaction chromatography. Conceivably, the observed decrease in PKC activity after OH-Tam treatment was due to an irreversible modification of PKC. Under the same conditions, OH-Tam did not inactivate purified cAMP-dependent protein kinase (holoenzyme) either in the presence or absence of cAMP, even at a high (100 M) concentration.
The Ca 2ϩ needed for this inactivation was above 50 M and optimal at 0.8 -1 mM in the absence of other regulators (data not shown). In the presence of phosphatidylserine and diolein, the concentrations of Ca 2ϩ required to promote this inactivation were lower (Fig. 10). In contrast, PMA either in the presence or in the absence of phosphatidylserine promoted this Tam-mediated inactivation of PKC even in the absence of Ca 2ϩ (Fig. 10). The inactivation of PKC induced by OH-Tam was found to be temperature-and time-dependent. The inactivation was very slow at 4°C and was rapid at 37°C. This inactivation of PKC also occurred with highly purified homogeneous preparations of PKC that lack any peroxidase activity. Among the Ca 2ϩ -dependent isoenzymes (␣, ␤, and ␥) tested, ␤-isoenzyme was more sensitive to the inactivation by OH-Tam with an IC 50 of 30 M (Fig. 11). In contrast, ␣ and ␥ isoenzymes were less sensitive to inactivation induced by OH-Tam and the IC 50 was approximately 120 M.
Identification of PKC Domain That Is Sensitive to OH-Tam-Previous studies suggested that Tam-induced inhibition of PKC caused by binding of Tam to both the catalytic and regulatory domains (15, 16, 19 -21). To identify the site that was irreversibly affected by OH-Tam, the Ca 2ϩ /phospholipid-independent activity of the enzyme using protamine sulfate, an indicator of the catalytic domain function independent from the regulatory domain, was determined. The protamine phosphotransferase activity was also lost parallel to Ca 2ϩ /lipid-dependent histone H1 phosphotransferase activity. Furthermore, among the cofactors tested, Mg 2ϩ enhanced OH-Tam mediated inactivation, while ATP/Mg 2ϩ complex protected the enzyme from this inactivation (Fig. 12). Protection was also observed with H-7, an inhibitor that competitively binds at the ATPbinding site on the enzyme. Furthermore, the catalytic domain (so-called M-kinase) generated by trypsin digestion was also inactivated by OH-Tam with an IC 50 of 25 M (Fig. 13). This concentration was nearly 2-fold lower than that required with holoenzyme. However, the regulatory domain generated by trypsin digestion was inactivated by OH-Tam only at higher concentrations of OH-Tam (IC 50 Ͼ 200 M). Unlike the catalytic domain, the regulatory domain lost its sensitivity upon separation from the catalytic domain by proteolysis.
An attempt was made to determine whether OH-Tam binds  11. Differential susceptibility of purified Ca 2؉ -dependent isoenzymes of PKC to irreversible inactivation induced by OH-Tam. PKC isoenzymes, separated by hydroxylapatite chromatography, ␣ (type III), ␤ (type II), and ␥ (type I) and the unseparated mixture of all three isoenzymes (approximately 1 unit) treated with the indicated concentrations of OH-Tam as described in the legend to Fig. 9 and after removing the OH-Tam the PKC activity was determined.
to the same site(s) where Tam also binds or if OH-Tam has its unique binding site. The enzyme was incubated with OH-Tam (50 M) at the conditions that favor inactivation, and, in another set of samples, OH-Tam was coincubated with a 4-fold excess of Tam to determine whether or not Tam could block OH-Tam action. Nearly 50% of the PKC activity was inactivated with OH-Tam alone, while with Tam coincubation, OH-Tam inactivated only 18% of the kinase activity. This suggested that both Tam and its metabolite OH-Tam would bind to the same site(s) on the PKC. Tam induced a reversible effect on the enzyme, while OH-Tam induced an irreversible modification of the enzyme leading to an inactivation of the kinase.
To gain insight into the mechanism by which OH-Tam can affect PKC, we have tested antioxidant systems to block inactivation of PKC induced by OH-Tam. Mercapto agents, DTT (1 mM) or 2-mercaptoethanol (10 mM), significantly decreased this inactivation when they were present during incubation with OH-Tam (Fig. 14). However, once the enzyme was inactivated by OH-Tam, these thiol agents did not reverse this modification. Vitamin E, SOD, and catalase all partially blocked the inactivation of PKC induced by OH-Tam. Heat-inactivated SOD and catalase had no protective effect. This suggested that reactive oxygen species might have been formed during the incubation of PKC with OH-Tam, which were scavenged by these antioxidant systems. The protection offered by SOD and catalase suggests the formation of superoxide and hydrogen peroxide. The inhibition by vitamin E also suggests a possibility for generation of phenoxyl radical from the phenolic compound.
Importance of Vicinal Thiols in the Catalytic Domain to Sensitize PKC for the Inactivation Induced by OH-Tam-Previous studies have shown that phenoxyl radicals that formed from the phenolic compounds could react with thiols to produce reactive oxygen species (46,47). To determine whether vicinal thiols present within the catalytic domain could play a role in mediating the phenolic compound or phenoxyl radical-mediated inactivation of PKC, the enzyme was initially treated with a nitric oxide-generating agent, S-nitrosocysteine, to mask the vicinal thiols by inducing the formation of disulfide bond(s) and then treated with OH-Tam. The treatment with S-nitrosocysteine resulted in a modification of the enzyme with a loss of PKC activity, which was reversed by a treatment with 10 mM DTT (Fig. 15). In contrast, the modification of PKC induced by OH-Tam was not reversed by DTT. Nonetheless, the pretreatment with S-nitrosocysteine and subsequent treatment with OH-Tam resulted in a lack of DTT-resistant inactivation of PKC. 3 This suggested that masking the vicinal thiol groups present within the catalytic domain was sufficient to prevent the irreversible inactivation of PKC induced by OH-Tam. 3 It is important to remove S-nitrosocysteine by centrifuge column technique before incubation with OH-Tam, which otherwise resulted in inactivation of more enzyme. Nitric oxide released from S-nitrosocysteine reacted with superoxide released from the incubation with OH-Tam to form a more reactive oxidant (presumably peroxynitrite), which inactivated the enzyme. The PKC activity remained after incubation with OH-Tam in the presence of these antioxidants was expressed as the percentage of the activity that observed with the appropriate controls carried out with the antioxidant in the absence of OH-Tam. DISCUSSION Although higher (Ͼ100 M) concentrations of Tam were required to inhibit PKC in a reversible manner in the test tube, with intact cells, it required substantially lower (5-20 M) concentrations to induce an initial membrane translocation, subsequent irreversible activation, and finally irreversible inactivation of this enzyme. By initially partitioning in the membrane, it is possible that Tam may inhibit PKC in a reversible manner in intact cells at a lower concentration than that required in the test tube. However, as suggested by these studies, additional complementing mechanisms of action of Tam could induce a bidirectional regulation of PKC with Tam at lower concentrations. This is the first report showing the role of oxidative stress in mediating the Tam effects on PKC as well as cell growth inhibition. Initially, Tam-related agents by partitioning into the membrane may induce local effects. The stabilization of the bilipid layer by Tam similar to cholesterol has been suggested previously by others (48). A decrease in membrane fluidity by Tam was shown previously (49). These membrane effects of Tam may trigger transmembrane signal transduction events and oxidative stress. This thesis was supported based on the following four key observations in this study. First, there is a good correlation with Tam-mediated effects on PKC and the release of arachidonic acid. Second, all the early cellular effects of Tam including the release of arachidonic acid, PKC translocation, irreversible activation and down-regulation of PKC, and cell growth inhibition were all inhibited by a variety of antioxidants. Third, the Tam-induced inhibition of cell growth was blocked efficiently by antioxidant vitamin E. Finally, a metabolite of Tam, OH-Tam, unlike the parent drug, induced an irreversible inactivation of purified PKC, which was partially prevented by various antioxidants.
The unique structural aspects of PKC may make it a suitable candidate for bidirectional regulation induced by oxidants (24). A selective oxidative modification of the regulatory domain of PKC results in a generation of Ca 2ϩ /lipid-independent form of the enzyme, while the modification of the catalytic domain leads to an inactivation of the enzyme (24). The regulatory domain contains 12 cysteine residues, which coordinate the binding of 4 zinc atoms (50), and the zinc-thiolate structure is required for binding of phorbol ester and diacylglycerol (51). This positively charged zinc-thiolate structure is more susceptible to oxidative modification by anionic oxidants than that of free thiolates present within the catalytic domain (24). Such differences in the reactivity of the thiolates in PKC can lead to the activation of the enzyme when the oxidants are generated at lower concentrations, and an inactivation of the enzyme by a modification of thiols in the catalytic site with an increase in the generation of oxidants.
Oxidants have been shown to activate both phospholipases A 2 and D, which can release directly or indirectly arachidonate from the membrane phospholipids (52,53). In a recent study, an activation of phospholipase D by micromolar concentrations of Tam in intact cells was reported (54). Several agents, such as nonphorbol tumor promoters, staurosporine, thapsigargin, okadaic acid, and calyculin-A, that promote the release of arachidonic acid have been shown to induce a cytosol-to-membrane translocation of PKC (55,56). Once PKC is associated with the membrane, its susceptibility to oxidative modification may be enhanced (24). A lack of inactivation of protein kinase A under these conditions suggests certain specificity in the effects of Tam on protein kinases in intact cells.
The metabolite OH-Tam is the major circulating species formed by the hepatic biotransformation of Tam in rats (1). However, in humans, OH-Tam is a minor metabolite, and it is not known whether it is formed in sufficient concentrations in the Tam-treated cells in culture. Since Tam is a reversible inhibitor of PKC in the test tube while it is an irreversible inactivator in intact cells, the observed irreversible inactivation of PKC in the test tube by OH-Tam suggests at least one of the metabolites formed from Tam in the body is capable of irreversibly inactivating PKC. Although we are sure that PKC inactivation in the Tam-treated cells is initiated by an oxidative process, it is not clear at present whether this inactivation process is mediated directly by a metabolite formed from Tam or mediated by endogenous oxidants generated in response to Tam action. In spite of the fact that OH-Tam was a catalytic site-directed inactivator of PKC, it did not inactivate the catalytic subunit of PKA, suggesting that certain specificity may exist in the action of OH-Tam to affect limited protein kinases.
Multiple mechanisms leading to formation of oxidants may be involved in mediating the inactivation of PKC induced by Tam in the cell or OH-Tam in the test tube. One possibility is that an initial formation of phenoxyl radical could react with vicinal thiols to produce reactive oxygen species that can inactivate PKC (46,47). The other possibility is that autoxidation of certain phenolic agents such as hydroquinone and catechol could produce superoxide and hydrogen peroxide through the redox cycle process (57). Alternately, Tam may form a carboncentered radical and participate in inducing oxidative stress. Potential formation of carbon-centered radicals from related agents such as triphenylmethane was discussed before by others (30). Although in this study, a prooxidant role of Tam or OH-Tam has been presented, previous studies have suggested an antioxidant function for these agents (48). It is possible that depending on the conditions, Tam or its hydroxymetabolite could function as either prooxidant or antioxidant. This is not an unusual situation, since several other phenolic agents have previously been shown to influence oxidative processes in a bimodel manner depending on the conditions (58, 59).
The pharmacological interest of estrogen-related agents had originally began with the observation of estrogen-like effects along with 1 mM CaCl 2 was added and the samples were incubated for 10 min. Then the low molecular weight compounds present in the treated PKC samples were removed by the centrifuge column technique. Then the treated PKC samples were subdivided into two aliquots, and to one aliquot DTT was added to 10 mM final concentration. PKC activity present in the both aliquots (with or without DTT) was determined.
produced by stilbene and triphenylethylene. Although the role of redox regulation and the formation of reactive oxygen species have been well documented for a stilbene derivative, diethylstilbesterol (60), redox metabolism was not studied for the metabolites of triphenylethylene derivative, Tam. Similarly, it has been well established that the carcinogenic effects of high concentrations of estradiol may involve a formation of catechol estrogens and reactive oxygen species (61). The oxidants play an important role in tumor promotion (62)(63)(64)(65). Furthermore, drugs such as chloroquine induce retinopathy, which may involve its ability to produce oxidative stress (66). Therefore, the oxidative stress induced by Tam may have some role in producing its side effects, such as increased incidences of uterine cancer and retinopathy in humans.
In ER-positive cell lines, Tam can elicit estrogen-reversible growth inhibitory effects at a concentration range of 10 -100 nM (4 -7). However, at such a concentration range, Tam in ERpositive cell line (MCF-7) growing in a regular serum-containing medium did not affect PKC within a short period of time (2 h). It is difficult to interpret the changes occurring in PKC after a prolonged treatment with Tam at lower concentrations. In order to sensitize the ER-positive cell lines to submicromolar concentrations of Tam, it is necessary to grow these cells for a few days in a phenol red-free medium supplemented with dextran-coated charcoal-treated serum. Therefore, further studies in this direction are certainly needed. These studies do not exclude the role for ER in the action of Tam in ER-positive cell lines. The Tam-induced oxidative stress may complement the effects occurring through ER. Both estrogen and Tam were reported to induce peroxidase activity in target tissues such as uterus and mammary gland (67). Phenol red is known to mimic certain actions of estrogen in breast carcinoma cells in culture (68). This dye is also a good substrate for peroxidase (69). Moreover, peroxidase has been recently shown to activate Tam yielding covalent protein adducts (70).
Originally, it was thought that Tam inhibits growth of only ER-positive cells. Subsequently it was shown to inhibit growth of other cell types which lack ER (8 -10). In a recent study, it was shown that A549 lung carcinoma cells that lack ER were inhibited by Tam at nanomolar concentrations (11). Furthermore, Tam inhibits growth of MCF-7 breast carcinoma cells even in the absence of estrogen or phenol red in the medium (6,9). Breast carcinoma in humans that initially responded to Tam therapy, subsequently develops a resistance to this drug (10). From Tam-sensitive MCF-7 breast carcinoma cells, Tamresistant sublines were cloned (71). The presence of ER in the breast carcinoma cells that are resistant to Tam (71) suggests that ER alone is not sufficient to mediate Tam-induced growth inhibitory effects, and it could be possible that other mechanisms may complement the action of ER in inducing cell growth inhibition. Recent studies revealed that mutations in ER appeared in only a small percentage of tumors that are resistant to Tam and may not alone contribute to the development of resistance to Tam (72). Based on the current study, it is possible that a cellular resistance to Tam could develop as a consequence of multiple factors, which may include a decrease in metabolism of the Tam, a low rate of generation of oxidants in response to Tam, and an increase in expression of antioxidant systems.
Tam also resembles phorbol ester to some extent by inducing membrane translocation and down-regulation of PKC. Simply an induction of these changes alone may not lead to tumor promotion. Bryostatin can induce PKC membrane translocation and down-regulation, but can also block some actions of PMA and has cancer therapeutic potential (73,74). Nevertheless, bryostatin differed from TPA in inducing the extent of translocation and down-regulation of various isoenzymes (74). Such quantitative differences may explain the action of tumor promoter versus a chemopreventive agent.
Further studies in vivo are certainly required to understand the relation between Tam-induced oxidative regulation of PKC and the organospecificity involved in therapeutic action of this drug or its unwanted side affects such as induction of uterine and liver cancer or retinopathy. In the event that these ERindependent oxidative changes in PKC are related to side effects of these drugs, antioxidants, particularly of dietary origin such as vitamin E, vitamin C, and ␤-carotene, may be a beneficial addition to Tam therapy to reduce the risk of side effects produced by Tam.