Tamoxifen-induced growth arrest and apoptosis in pituitary tumor cells in vitro via a protein kinase C-independent pathway

Cancer Letters 185 (2002) 131–138 www.elsevier.com/locate/canlet

Tamoxifen-induced growth arrest and apoptosis in pituitary tumor cells in vitro via a protein kinase C-independent pathway Marie Simard a, Wei Zhang a, David R. Hinton b,c, Thomas C. Chen b, Martin H. Weiss b, Yu-Zhuang Su b,c, Rayadu Gopalakrishna d, Ronald E. Law e, William T. Couldwell a,* a Department of Neurosurgery, New York Medical College, Valhalla and New York, NY, USA Department of Neurological Surgery, University of Southern California School of Medicine, Los Angeles, CA, USA c Department of Pathology, University of Southern California School of Medicine, Los Angeles, CA, USA d Department of Cell and Neurobiology, University of Southern California School of Medicine, Los Angeles, CA, USA e Division of Endocrinology, Diabetes and Hypertension, Department of Medicine, University of California Los Angeles, Los Angeles, CA, USA b

Received 17 August 2001; received in revised form 24 April 2002; accepted 6 May 2002

Abstract Protein kinase C (PKC), a kinase family involved in cell signal transduction, is overexpressed in most pituitary adenoma cells. We studied the effect of tamoxifen, an estrogen receptor antagonist and also a protein kinase inhibitor, on pituitary tumor cell proliferation and the induction of apoptosis; and we compared its effects with those of another PKC inhibitor, staurosporine. Tamoxifen induced growth arrest and apoptosis in a mouse pituitary adenoma cell line, AtT20, and in low-passage human primary pituitary tumor cell cultures. Staurosporine also inhibited pituitary tumor cell growth. PKC activity in AtT20 cells was inhibited by staurosporine and by prolonged treatment with phorbol myristate acetate, which down-regulates PKC activity, but not by tamoxifen, at the dosages used to induce apoptosis. Our findings suggest that tamoxifen induces apoptosis in AtT20 cells independent of a classical PKC isozyme pathway. q 2002 Elsevier Science Ireland Ltd. All rights reserved. Keywords: Apoptosis; Pituitary adenoma; Protein kinase C; Tamoxifen; Staurosporine

1. Introduction Protein kinase C (PKC) is an intracellular signal transduction system consisting of a family of serine/ threonine-specific kinases, which are activated differentially by Ca 11, diacylglycerol, and phospholipids. At least 11 subspecies have been identified: the conventional or calcium-dependent (a, b, g), the novel or calcium independent (d, 1, h, u, m), and the * Corresponding author. Department of Neurosurgery, University of Utah, Suite 3B409, 30 North 1900 East, Salt Lake City, UT 84132-2303, USA. E-mail address: [email protected] (W.T. Couldwell).

atypical (z, l) isozymes [1,2]. Upon cell stimulation, the enzyme undergoes translocation from the cytosol to the plasma membrane, which is a hallmark of PKC activation. The multiple isoforms display characteristic subcellular localization in different cell types and may participate, according to their enzymological properties, in various cellular processes including growth and differentiation [2,3]. Established rat pituitary adenoma cells and surgically resected frozen human adenoma specimens express high levels of PKC activity compared to non-transformed adenohypophysis [4,5]. The relative expression of the soluble PKC subspecies differs between normal and adenomatous pituitary cells, with PKC a being predomi-

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nantly expressed in pituitary adenomas [6,7]. PKC activity is greater in invasive than in non-invasive adenoma cells [4,5] and thus may modulate the proliferation rates and invasive phenotype(s) of these tumors. Pituitary adenoma cell proliferation rates are sensitive to inhibitors of the PKC system [4,8,9]. The kinase inhibitors staurosporine and tamoxifen reduce [ 3H]thymidine uptake in pituitary adenoma cells [4], and tamoxifen induces apoptosis in GH3 pituitary adenoma cells [10]. However, their mechanisms of action remain unclear. We examined the effects of tamoxifen on cell proliferation and induction of apoptosis in an established rodent pituitary adenoma cell line, AtT20, and in five human low-passage pituitary tumor cultures in vitro. PKC activity was measured in AtT20 cells treated with or without tamoxifen and compared with PKC activity in cells treated with other PKC modulators. Our observations indicate that although growth arrest and apoptosis can be induced by inhibition of PKC, tamoxifen appears to inhibit pituitary tumor cell growth and induce apoptosis via a PKC-independent pathway.

2. Materials and methods 2.1. Cell cultures and chemicals The mouse pituitary adenoma cell line AtT20 was obtained from American Type Culture Collection (ATCC, Rockville, MD). Six human pituitary tumor primary cell cultures were derived with a standard method [8] from surgical specimens obtained from patients at the University of Southern California University Hospital, CA (a 21-year-old male with a null-cell non-invasive macroadenoma, a 46-year-old female with a growth hormone (GH)-secreting pituitary adenoma, a 51-year-old male and a 61-year-old male with non-secreting pituitary adenomas, and a 21year-old male with a pituitary carcinoma) and at Westchester Medical Center, NY (a 36-year-old male with a GH-secreting macroadenoma). Pituitary cultures were considered adequate if more than 95% of the cells in culture were of adenohypophysial origin, as characterized morphologically by a neuropathologist (D.R.H.). Cells were grown in Dulbecco’s

modified Eagle’s medium (DMEM) supplemented with 10% fetal bovine serum (FBS), penicillin–streptomycin (100 units/ml and 100 mg/ml, respectively) and 10 mM HEPES buffer (pH 7.0) at 378C in a humidified 5% CO2 incubator, routinely tested for mycoplasma contamination, and studied after low passage (two to three passages). All medium constituents were purchased from Life Technologies (Grand Island, NY). The PKC modulators included staurosporine (Calbiochem, La Jolla, CA), tamoxifen, and 4-phorbol-12-myristate-13 acetate (PMA) (both from Sigma, St. Louis, MO). The green nucleic acid dye, Sytox, and Slow Fade were purchased from Molecular Probes, Eugene, OR. All other chemicals, such as 3(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT), polylysine, and dimethyl sulfoxide (DMSO) were from Sigma. 2.2. Cell viability assay Pituitary tumor cell growth and viability were evaluated by the microculture tetrazolium (MTT) assay, in which the cell number was quantitated by the amount of tetrazolium dye reduction [11], as previously described [12]. In brief, cells were seeded at a density of 2 £ 10 3 cells/well in 100 ml of 10% FBS–DMEM in flat-bottom 96-well plates (Corning Glass Works, Corning, NY). The test agents, tamoxifen, which dissolves in ethanol, and staurosporine, which dissolves in DMSO, were added to wells containing cells at predetermined concentrations in replicates of five. Control cultures in replicates of five wells were treated with vehicle solution containing 0.1% ethanol or 0.1% DMSO, respectively. After 48 h, MTT at a concentration of 5 mg/ml was added to each well, and the plates were incubated for 4 h. After formation of the formazan crystals, the culture medium supernatant was removed from the wells without disruption of the formazan precipitate. The formazan crystals were dissolved in 100% DMSO (150 ml/well). Absorbance was measured at 570 nm with a microplate spectrophotometer (Dynatech MR700; Chantilly, VA) interfaced with a Macintosh computer. 2.3. Protein kinase C assay Classical PKC can be activated by 1,2-diacylglycerol or its surrogate, PMA, and various stimuli and exhibits altered subcellular distribution by association

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with plasma membranes and nuclear and cytoskeletal components [1]. Furthermore, PMA, which prolongs the association of PKC with the membrane, can initiate the degradation of the PKC molecule and its sustained disappearance from the cells. Thus, the cytosolic and particulate fractions of PKC were isolated after the cells had been treated with tamoxifen, staurosporine, PMA, or their respective controls (0.1% ethanol or 0.1% DMSO) and separated by diethylaminoethyl (DEAE)-cellulose chromatography as previously described [13]. Briefly, the soluble and detergent-solubilized membrane fractions were isolated from cell extracts and were applied to a 1ml DEAE-cellulose (DE-52) column previously equilibrated with buffer A (20 mM Tris–HCl, pH 7.5/ 1 mM ethylenediaminetetraacetate/0.1 mM DTT). After the column was washed with 4 ml of buffer A, the bound PKC was eluted with 2.5 ml of 0.1 M NaCl in buffer A. The enzyme fractions were stored at 2708C before assay for PKC. Classical PKC isozyme (a, b, g) activity was determined with the established method by measurement of adenosine triphosphate (ATP) transfer into lysinerich histone using mutiwell plates with fitted filtration discs as described previously [13]. In brief, the substrate and the metal-ion components necessary for the PKC assay (50 mM Tris–HCl, pH 7.5/25 mM MgCl2/0.8 mM CaCl2/0.2 mM ATP/histone (0.25 mg/ ml)/0.1 mM leupeptin) were premixed initially and stored at 2208C. This substrate/metal-ion mixture then was thawed, and [ 32P]ATP was added to it to bring the 32P radioactivity to approximately 1.5 million cpm per 50 ml (120 cpm/pmol ATP) of the reactive mixture. For the PKC assay, 96-well plates with fitted polyvinylidene difluoride membrane filters (Durapore, pore size 0.45 mm) were used. Samples (25 ml) to be assayed for PKC activity were pipetted into each of four wells. Fifty microliters of 20 mM EGTA was then added to the first two wells, and 50 ml of sonicated lipid mixture (100 mg/ml of phosphatidylserine and 10 mg/ml of diolein) was added to the other two wells. The reaction was initiated by the addition of the prepared substrate/metal-ion mixture (50 ml) to all wells containing PKC samples, and the plates were incubated at 308C for 5 min. To terminate the reaction, 75 ml of 70% trichloroacetic acid (TCA) was added to each well. The plates were gently shaken for 5 min at 48C and all TCA was removed by

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vacuum. After being washed six times with 200 ml of ice-cold 25% TCA and dried under a heating lamp for 5 to 10 min, the filters were punched into scintillation vials that contained 4 ml of scintillation cocktail and examined in a scintillation counter (Beckman). PKC activity was determined by the count of radioactivity collected from precipitated samples bound to histone-associated 32P and was expressed as units/mg soluble protein, where 1 unit of enzyme transfers 1 nmol of phosphate to histone H1 per min at 308C. For each cell culture condition, three separate cultures were assayed. 2.4. DNA fragmentation assay and immunochemistry Pituitary tumor cell apoptosis was determined with a DNA fragmentation assay as described previously [14]. The cells were treated for up to 48 h with tamoxifen (1–to 20 mM), PMA (1 mM), or their respective controls (0.1% ethanol and 0.1% DMSO). DNA concentration was determined by A260 measurements with a Beckman DU-640 spectrophotometer. Equal amounts of DNA were electrophoresed on a 1.2% agarose gel containing 0.5 mg/ml ethidium bromide and visualized by UV fluorescence. In addition, tamoxifen-induced cell death was examined by immunohistochemistry to demonstrate apoptosis. The cells from a GH-secreting pituitary adenoma were seeded onto 12-mm diameter glass coverslips coated with 10% polylysine, placed into 24-well cell culture plates (5 £ 10 4 cells/well), and incubated at 378C for 24 h. The cells were then treated with 10 mM of tamoxifen or with a vehicle solution containing 0.1% ethanol. After a 24-h incubation, the cells were fixed in 4% paraformaldehyde for 10 min, permeabilized with 0.1% Triton X-100, and incubated with the single step dead cell indicator Sytox (1 mM) for 30 min at room temperature. After the cells were washed with phosphate-buffered saline several times, the coverslips were mounted in Slow Fade. Immunofluorescence was visualized with a Bio-Rad MRC 1000 confocal scanning microscope (Hercules, CA). 2.5. Statistical analysis All comparisons between groups were performed with a one-way analysis of variance (ANOVA), with the Student–Newman–Keuls method for post hoc pair

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wise multiple comparisons to detect differences between individual group means.

3. Results 3.1. Dose-dependent effects of tamoxifen and staurosporine on the growth of pituitary tumor cells An MTT assay was performed after treatment of the AtT20 and several low-passage pituitary tumor cell lines with tamoxifen or staurosporine. Over the 48-h treatment period, tamoxifen in 5–10 mM concentrations significantly inhibited the growth of AtT20 cells and in 15–20 mM concentrations significantly inhibited the growth of four low-passage human pituitary adenoma cell cultures in a dose-dependent fashion compared to control cells treated with ethanol (Fig. 1). A low-passage human pituitary adenocarcinoma cell culture was sensitive to growth inhibition by treatment for 48 h with tamoxifen at doses of 1– 5 mM (Fig. 1). After treatment for 48 h, staurosporine at a concentration of 1 mM significantly inhibited the growth of AtT20 cells and at concentrations of 0.1– 1 mM inhibited the growth of three low-passage human pituitary adenoma cell cultures in a dosedependent fashion (Fig. 1). 3.2. Time-dependent modulation of PKC activity in AtT20 cells by PMA and staurosporine, but not by tamoxifen Classical PKC isozyme activity was determined in AtT20 cell cultures treated for up to 48 h with tamoxifen, staurosporine, and PMA. Treatment with tamoxifen (10 mM) for 48 h failed to inhibit PKC activity in these cells compared to the 0.1% ethanol-treated control cells (Fig. 2A). In contrast, PKC activity was reduced within 48 h after treatment with staurosporine (10 nM) compared to 0.1% DMSO vehicle solution (Fig. 2B). Treatment of AtT20 cells with PMA, compared with 0.1% DMSO vehicle solution, initially increased membrane-bound activity at 24 h, consistent with translocation of the enzyme from the cytosol to the membrane. At the 48-h time point, this PKC activity had markedly decreased (Fig. 2C), demonstrating that the PKC activity in AtT20 cells is sensitive to PMA.

3.3. Induction of apoptosis in AtT20 cells and in lowpassage primary pituitary tumor cells by tamoxifen After treatment with 10 mM tamoxifen for 48 h, the AtT20 cells demonstrated a classic ladder pattern of oligonucleosomal-sized fragmented DNA indicative of late apoptosis (Fig. 3A, lane 3). DNA laddering of low-passage human pituitary tumor cells also occurred after treatment with tamoxifen, 20 mM (Fig. 3B, lane 4), and was absent after treatment with vehicle solution (Fig. 3B, lane 1). If the induction of apoptosis depended on PKC activity, prolonged treatment of cells with PMA should also initiate apoptosis in this assay. As predicted, treatment of the AtT20 cells and a lowpassage human pituitary adenoma culture for 48 h with 1 mM PMA, which down-regulates PKC activity, induced DNA fragmentation (Fig. 3A, lane 4, and Fig. 3B, lane 5). In addition, staurosporine at concentrations of 10 and 100 nM similarly induced apoptosis in the AtT20 cells (data not shown). To further characterize the induction of apoptosis by tamoxifen, immunohistochemical labeling against nuclear DNA in GH-secreting human pituitary adenoma cells in culture was performed. Treatment of the cells with tamoxifen (10 mM, 24 h) induced nuclear fragmentation (Fig. 4B) in comparison to control cells treated with vehicle solution (Fig. 4A).

4. Discussion 4.1. Protein kinase C: a potential target for inhibition of pituitary adenoma cells We observed elevated basal PKC activity for the classical calcium-dependent PKC subspecies in the AtT20 cells. As demonstrated in Figs. 3A,B, treatment of the established cell line AtT20 and of a lowpassage primary adenoma culture with as little as 1 mM PMA for 48 h (a time period shown to downregulate the classical PKC isozyme activity in these cells) induced apoptosis, supporting an active role for PKC in this process. The high PKC activity may prevent the execution of a latent cell death program. Moreover, staurosporine, which acts at the catalytic domain of PKC [15], down-regulated total classical PKC activity (Fig. 2B), inhibited cell proliferation

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Fig. 1. Antiproliferative and cytotoxic effects of tamoxifen and staurosporine on the mouse pituitary adenoma cell line AtT20 and low-passage human pituitary tumor cell cultures. Cultured cells were treated with tamoxifen and staurosporine at increasing concentrations, or with their respective controls (0.1% ethanol and 0.1% DMSO), and the number of viable cells was determined with MTT as described in Section 2. Data shown (mean ^ standard error, n ¼ 4) are typical of two independent experiments that yielded similar results. (**, Significantly different from respective control value, P , 0:01).

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(Fig. 1), and induced apoptosis (data not shown) in AtT20 cells. Similarly, staurosporine inhibited cell proliferation in several primary pituitary adenoma cell cultures (Fig. 1). These findings suggest a potential target of PKC for inhibition of the proliferation of pituitary tumor cells. However, the activity of particulate PKC in AtT20 cells was not inhibited significantly by staurosporine. Although the reason is not known, the pattern of staurosporine inhibition of PKC in pituitary tumor cells might be different from that in other cell types, such as glioma cells. 4.2. Tamoxifen: inhibition of cell growth and induction of apoptosis via a classical PKC isoformindependent pathway Our results demonstrate that tamoxifen inhibits the growth of pituitary adenoma cells in vitro. Treatment

Fig. 2. Protein kinase C activity in the mouse pituitary adenoma cell line AtT20 after treatment with PKC modulators. PKC activity was determined in control AtT20 cell cultures treated with vehicle solutions and those treated with the PKC modulators. Treatment with tamoxifen (10 mM) over a 48-h time interval failed to inhibit PKC activity in AtT20 cells compared to control cells treated with a 0.1% ethanol solution (A). PKC activity decreased significantly over the same interval after treatment of the cells with 10 nM staurosporine compared with control cells treated with a 0.1% DMSO solution (B). Treatment of the AtT20 cells with the phorbol ester PMA (1 mM) initially produced a translocation of the enzyme to the membrane fraction at 24 h, followed by a reduction in total PKC activity at 48 h consistent with previous reports of down-regulation (C). All values are the mean ^ standard error of triplicate assays. Total PKC activity is the sum of the particulate and cytosolic fractions.

Fig. 3. Induction of DNA fragmentation in AtT20 and low-passage human pituitary adenoma cells by tamoxifen. Treatment of adenoma cells with tamoxifen and DNA isolation are described in Section 2. In (A), DNA isolated from AtT20 cells treated with 0.1% ethanol solution (control) (lane 1), and treated with 5 and 10 mM tamoxifen (lanes 2 and 3) for 48 h were electrophoresed in 1.2% agarose and stained with ethidium bromide. Note the presence of oligonucleosome-sized DNA fragments from cells treated with 10 mM tamoxifen, producing a classic ladder pattern in lane 3. Treatment of the non-functional human pituitary adenoma cells with tamoxifen (1–20 mM) for 48 h (B, lanes 1–4) also resulted in the production of oligonucleosomal DNA fragments at the higher dosage of 20 mM (B, lane 4). Treatment of the established AtT20 cell line and the primary pituitary cell culture with PMA induced DNA fragmentation after the same 48-h treatment period that produced down-regulation of PKC activity. PMA, at a dose as low as 1 mM, induced apoptosis in these cells after the 48-h treatment period (A, lane 4, and B, lane 5).

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Fig. 4. Tamoxifen-induced apoptosis in human pituitary adenoma cells. Immunodetection with Sytox green nucleic acid stain demonstrates DNA fragmentation in a primary human GH-secreting pituitary adenoma after treatment with tamoxifen, 10 mM, for 24 h (B); control cells were treated with a 0.1% ethanol solution (A).

of AtT20 cells with tamoxifen at doses of 1–10 mM failed to inhibit the activity of the classical PKC isoforms (Fig. 2A) despite inhibiting cell proliferation (Fig. 1) and inducing apoptosis (Fig. 3A). Taken together, these observations suggest that alternative mechanism(s) are responsible for the cytocidal effect of tamoxifen in this cell line. We cannot exclude the possibility that these effects may be mediated by inhibition or activation of one or several of the other PKC isozymes not measured in our assay. In addition to its known inhibition of estradiol-stimulated activation of estrogen receptors (ERs), tamoxifen exerts genomic effects in both hormone-responsive and hormone-independent tumors and affects growth factors and receptors considered to play an important role in the regulation of tumorigenesis [16–19]. The overexpression of c-myc [20], the antagonism of calmodulin [21], the modification of lipid second messenger formation [22], and alterations of cytoskeletal and nuclear lamins [23] are also considered to play key roles in determining nuclear apoptotic changes caused by tamoxifen. 4.3. Potential clinical implications Tamoxifen has been shown to slow the growth of a metastatic prolactin-secreting pituitary carcinoma that had failed to respond to conventional therapy [24]. The doses of tamoxifen necessary for the inhibition of

growth and the induction of apoptosis in the established AtT20 cell line and in a low-passage human pituitary adenocarcinoma culture are in the same concentration range as those reported for malignant gliomas [14]. Such therapeutic levels can easily be achieved with high-dose oral administration without dose-limiting toxicity [25]. Previously we measured tamoxifen and its active metabolite N-desmethyltamoxifen in glioma tissues from a patient treated with high-dose tamoxifen (160 mg/day); tissue concentrations reached 2.6 and 11 mM, respectively [26]. In a recent report, high-dose tamoxifen was used to treat patients with metastatic melanoma [27]. Serum tamoxifen levels were found to correlate with the dose administered, with a mean ranging from 0.9 mM at the 40mg dose to 4.6 mM at the 320-mg dose. This study suggests that tamoxifen possesses cytocidal activity that does not appear to be mediated via the classical PKC isozyme pathway(s). Tamoxifen, therefore, may have added therapeutic potential for use in combination with PKC inhibitors and may offer promise to patients with pituitary tumors for whom conventional therapies have failed.

Acknowledgements This work was supported by grants form the Amer-

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