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PLA2Activity Regulates Ca2+Storage-Dependent Cellular Proliferation
EXPERIMENTAL CELL RESEARCH ARTICLE NO.
244, 310 –318 (1998)
EX984181
PLA2 Activity Regulates Ca21 Storage-Dependent Cellular Proliferation Michael J. Petr,* Thomas C. Origitano,† and Robert D. Wurster*,†,1 *Loyola University Medical Center, Stritch School of Medicine, *Department of Neuroscience and Physiology; and †Department of Neurological Surgery, Maywood, Illinois 60153
The objective of this study is to determine the role of arachidonic acid (AA) in cell proliferation by inhibiting AA synthetic enzyme phospholipase A2 (PLA2) and to determine its involvement in the role of the second messenger intracellular calcium (Ca21). Methods used to determine the effects on proliferation of cell cultures of primary meningioma and astrocytoma U373-MG included treatment with micromolar concentrations of PLA2 inhibitors 4-bromophenacylbromide and quinacrine. Effects of these drugs on proliferation were further investigated by the application of concentrations that inhibit growth by 50% while antagonizing these agents with AA replacement. Free cytosolic Ca21 was measured with the use of fluorescent dye Fura-2 during PLA2 agonist/antagonist studies. These Ca21 measurements were performed in the absence of extracellular Ca21 to identify the contribution of intracellular Ca21 sources. PLA2 inhibition resulted in decreased growth of cultured astrocytoma and meningioma cells in a dose-dependent manner in the micromolar range. This inhibitory effect was antagonized by the addition of AA. PLA2 inhibition caused an elevation of basal-cytosolic-free [Ca21] while depleting internal Ca21 stores. These Ca21 changes were also antagonized by the addition of AA. In conclusion, these results demonstrate that AA, a PLA2 enzyme product, is involved in regulating the growth rate of these cell types. The PLA2 pathway also regulates the maintenance of the internal Ca21 stores. Ca21 is known to be a growth-related intracellular second messenger. These results suggest that the growth regulatory functions of AA are mediated by Ca21-dependent mechanisms. © 1998 Academic Press Key Words: arachidonic acid; calcium; PLA 2 ; proliferation.
INTRODUCTION
Arachidonic acid and proliferation. Eicosanoids modulate many signal transduction mechanisms. Products of 1 To whom correspondence and reprint requests should be addressed at Loyola University Medical Center, Stritch School of Medicine, Physiology, Neurological Surgery, Building 102, 2160 South 1st Ave., Maywood, IL 60153. Fax: (708) 216-6308.
0014-4827/98 $25.00 Copyright © 1998 by Academic Press All rights of reproduction in any form reserved.
arachidonic acid (AA) metabolism themselves are also directly involved in intracellular signaling [1–5]. AA pathways have been altered experimentally in other cell types to demonstrate growth-related effects. A possible relationship exists between an increased AA metabolism and some aspects of proliferative behavior [6 –14]. In endothelium and vascular smooth muscle, for example, AA pathways have been selectively manipulated resulting in altered cell growth rates [15]. Eicosanoids, arachidonic acid. Arachidonic acid is the parent bioactive lipid member of eicosanoids consisting of a C20 hydrocarbon chain with four double bonds and can be both membrane bound and cytosolic. Spatially, eicosanoids freely cross cell membranes and act in an autocrine/paracrine capacity [16]. Temporally, stimulus-induced AA production occurs after short-term second messengers, such as inositol phosphates, but precedes the occurrence of long-term signal transduction events such as protein phosphorylations [17]. The eicosanoid cascade may be induced by Ca21dependent pathways (phosphatidylcholine substrate) and Ca21-independent pathways (phosphatidylethanolamine substrate) and may be mediated by phospholipase C (PLC)-inositol trisphosphate (IP3)-Ca21 or protein kinase C (PKC) activation of phospholipase A2 (PLA2) [18 –20]. Receptors may also be directly coupled to PLA2-associated G-proteins [21]. PLA2 in astrocytes may be activated by ATP via purinergic receptors linked to G-proteins [22]. In addition to the activation of PLA2, AA is produced by alternative pathways. PLC or phospholipase D (PLD) activation generates accessible diacylglycerol (DAG), a substrate for DAG-lipase or monoglycerol lipase resulting in the generation of AA [23]. Once cleaved from the membrane by PLA2 or other pathways, AA may be metabolized by cyclooxygenase and lipoxygenase enzymes to generate prostaglandins (PG) and leukotrienes (LT). These eicosanoid products may result in a variety of receptor-mediated effects. Understanding the eicosanoid-related cellular mechanisms offers promising insights into regulation of cellular proliferation. The major focus of this paper is eicosanoid-mediated mechanisms affecting regulation of proliferation. These eicosanoid-mediated effects are suspected to occur due to the link of these lipid
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species to alterations in calcium (Ca21)-mediated signal transduction. MATERIALS AND METHODS Cell culture, cell line characteristics of U-373-MG human astrocytoma. These cultured cells were the product of a primary tumor originating from a 61-year-old, blood type A1 caucasian male by B. Westmark [24] and was purchased from American Type Culture Collection (Rockville, MD). These cells were tumorigenic in nude mice forming grade III astrocytomas and producing glial fibrillary acidic protein (GFAP) [25]. The growth rate of these cells was effected by alteration of calcium (Ca21) metabolism [26]. Cell culture, cell line characteristics of primary meningioma. Fresh tissue was retrieved from the surgical field. Tissue was aseptically placed in Earl’s balanced salt solution (EBSS) with penicillin 10 K units/L, streptomycin 10 mg/L, and amphotericin B 25 mg/L (Sigma St Louis, MO). This tissue was mechanically reduced to 2 to 5-mm3 pieces in a laminar flow hood. Tumor particles were incubated for 24 h at 37°C in 20 ml of minimal essential medium (MEM) with 10% fetal bovine serum (FBS)(1). Tumor cell isolation was achieved with MEM1 supplemented with 1 mg/ml collagenase A (Boehringer Mannheim, Indianapolis, IN), and 100 ml of 1 mg/ml deoxyribonuclease (Sigma). After enzymatic digestion the tissue was forced through 60 (0.23 mm) and 150 (0.104 mm) steel mesh (Sigma). Suspended tissue and fluid were split into two 150-ml flasks (Falcon, England). Meningioma culture use was limited to the first 5 passages due to unreliable characteristics that may occur with later passages. Meningioma cultures maintain tumor characteristics in culture as determined by light and electron microscopy and immunohistochemical studies [27]. Each experiment was performed with as many as three different tumors as culture sources. No effort was made to specifically select for age, gender, or tumor location. All tumors and cultures were characterized by histopathology and immunocytochemistry as meningioma ranging from benign to anaplastic. Culture maintenance and growth. Cultures were grown in a humidified incubator at 37°C with gas concentrations of 5% CO2/95% air, and media was replaced every 3 or 4 days. Cells were harvested for experimentation by adding 0.25% trypsin (Sigma) and 50 mM ethylene glycol-bis(b-aminoethylether) N,N,N9,N9,-tetraacetic acid (EGTA) for 8 min. After trypsin and EGTA, effects were quenched by the addition of serum and dilution. Cells were separated from solution by centrifugation. Cells from confluent cultures were harvested for growth studies and reseeded in 35 3 10-mm culture dishes (Fisher Scientific, Pittsburgh, PA) at a density of 2.5 3 105 cells/dish for the astrocytoma and 1.5 3 105 cells/dish for the meningioma. Pharmacological treatment for growth. Stock solutions of 10 mM 4-bromophenacylbromide (4-BPB) (Sigma) were prepared in 70% ethanol and were diluted to final concentrations in 2-ml petri culture dishes. Final ethanol concentrations in cultures were less than 0.35%. Studies show no difference in growth using 0.35% ethanol verses water controls (data not shown). Quinacrine (Sigma) was prepared in a stock solution of 10 mM in double-distilled water. All stock solutions were stored at 4°C and protected from exposure to light. Concentrations ranging above and below the EC50 for each drug and cell type were tested for effects on growth. Reagent administration was initiated 24 h postseeding. Reagents and media were changed daily for 3 days, and growth measurements were made on Day 4. Cells were harvested by trypsinization and counted with a hemocytometer using trypan blue exclusion to determine a change in growth rate as compared to matched control independent of cytotoxicity. Growth inhibition by PLA2 inhibitors quinacrine and 4-BPB at half their maximal effective dose was antagonized with the addition of 5 mM AA. AA and the PLA2 inhibitors were replaced with serum-
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containing media twice daily for 3 days. Cells were then harvested and counted on Day 4. High performance liquid chromatography (HPLC) methods. Cells from four confluent 75-ml culture flasks were harvested by scraping. These cells were spun to a pellet and washed with 5 ml of Dulbecco’s buffer containing (in mM): 2.7 KCl, 1.5 KH2PO4, 137 NaCl, 8.1 NaH2PO4 z H2O, 2 glucose, and 1 CaCl2 at pH 7.4. Preparations were sonicated to rupture cells for a protocol similar to that used by Voltera et al. [28]. Sonicated cells were incubated with 1 mCi tritiated arachidonic acid (3H-AA) (210 Ci/mmol, 7770.0 GBq/mmol,) or 1 mCi tritiated phosphatidylcholine, L-a-1-stearoyl-2-arachidonyl [arachidonyl-5,6,8,9,11,12,14,15-3H (N)] (160.0 Ci/mmol, 5920.0 GBq/mmol) (3H-PC) (Dupont, NEN) at 37°C. The assay was terminated by the addition of 265 ml of 5 N formic acid and cooling on ice. Preparations were separated three times with ethyl acetate in a 1 to 1-vol ratio. The organic phase was then evaporated in a speed evacuation centrifuge. Dry lipid was redisolved in mobile phase B and loaded into the autoinjector. The aqueous phase was saved for protein analysis. Reverse-phase HPLC was performed by the method of Powell [29]. A gradient of polar mobile phase A to nonpolar mobile phase B was used with pumps and gradient mixer (Rainin). The gradient at 0 min was 100% mobile phase A (75% H 2 O, 25% CH 3 CN, 0.001% CF3COOH), 40 min mobile phase A was 20%, while mobile phase B was 80%, 60 –90 min mobile phase B was 100% (7.5% H2O, 54% CH3OH, 38.5% CH3CN, 0.001% CF3COOH). An autoinjector (Shamadzu), ultraviolet detector (Waters), and Flo-one/b scintillation radiodetector (Canberra-Packard) were connected in series. Cell numbers used for each experiment were standardized by normalization to protein. The ultraviolet detector was used to standardize elution times of a number of nonradioactive leukotriene samples such as LtB4, LtC4, 5HETE and 12HETE. Measurement of free intracellular calcium concentrations: Solutions and loading Fura-2-AM. Fluorescent Ca21 indicator Fura-2acetoxymethyl ester (Molecular Probes, Eugene, OR) was chosen for this study based on the published dissociation constant (Kd) (224 nM at 37°C and 135 nM at 22°C). These Kd values theoretically allowed accurate measurements of the free cytosolic Ca21 from resting values of 10 nM to the mM range. Fura-2-AM is lipophilic and not soluble in aqueous solutions, but does readily cross the lipid bilayer. Once the AM-ester crosses into the cytosol, nonspecific esterases cleave the ester, resulting in a charged free acid form which is not lost from the cell as rapidly as the parent lipid soluble compound [30]. Since AM-esters are insoluble in aqueous solutions, indicator was placed in micelles of the dispersing agent pluronic acid (Molecular Probes). Fura-2-AM was dissolved in dimethylsulfoxide (DMSO) (Sigma) at a concentration of 1 mg/ml for stock solution. The indicator preparation was added to Krebs-bovine serum albumin (BSA) 1 mg/ml solution resulting in an indicator end concentration of 1 mM [31]. Cell cultures were grown as a monolayer on polylysine-coated glass coverslips. These coverslips were placed in a 35 3 10-mm culture dish (Becton Dickenson, Lincoln Park, NJ). All serum was removed by washing with Krebs Ringer solution (KR). Coverslips containing cultured cells were covered with 1 ml of loading solution and were allowed to incubate for 1 h. Coverslips were then rinsed twice with KR and were ready for use. To prevent photo bleaching, all preparations with fluorescent indicators were used under conditions that provide protection from light [30]. Krebs Ringer solution contained: 125 mM NaCl, 1.2 mM KH2PO4, 1.2 mM MgSO4, 5 mM NaHCO3, 25 mM Hepes, 6 mM glucose (Sigma), 5 mM KCl, and 1.3 mM CaCl2 (Mallinckrodt, Paris , KE). Ca21- free KR was made as suggested above with the omission of CaCl2. The pH was adjusted to 7.4 with 1 N NaOH. Calcium measurement. Glass coverslips with the monolayer were placed in a cuvette with a perfusion apparatus. This cuvette was placed in the spectrophotometer chamber (Perkin Elmer, Buckinghamshire, England). Excitation wavelengths of 340 and 380 nm were used for Fura-2 bound and unbound to Ca21. Emission measure-
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FIG. 1. Dose dependent growth data are expressed as the mean and standard deviation of percentage of control. Solid squares (astrocytoma) and solid triangles (meningioma) demonstrate a dose-dependent inhibition of growth compared to untreated controls. (A) PLA2 inhibitor 4-BPB demonstrated an IC50 of 35 and 22 mM for astrocytoma and meningioma, respectively. (B) PLA2 inhibitor quinacrine demonstrated an IC50 of 6 and 2 mM for astrocytoma and meningioma, respectively. Growth curves are analyzed by one-way ANOVA and found to be statistically significant with P # 0.05, n 5 8.
ments in the 510 nm range were taken during fast frequency changes between excitation filters 340 and 380 nm to produce data points with comparable temporal relationships. Free-Ca21 concentrations were calculated with the Grynkiewicz equation [32].
way ANOVA and multiple comparisons. Data which have a P value # 0.05 were considered to be statistically significant.
RESULTS Free [Ca21]I nM 5 k d[(R 2 R min /R max 2 R)(b)]. Fluorescence scanning time was between 300 and 600 s which was enough time to measure intracellular changes due to positive controls such as acetylmethylcholine (MC) (5 mM) or cyclopiazonic acid (CPA) (30 mM). MC is a muscarinic-specific agonist which activates phospholipase C and inositol trisphosphate-induced intracellular Ca21 release from the endoplasmic reticulum [33]. CPA is a reversible inhibitor of the endoplasmic reticular Ca21ATPase [34]. Measurements were determined for control and experimental groups in order to determine the result of drug application on basal intracellular Ca21 ([Ca21]i) as well as agonist-stimulated cells. Effects of eicosanoid-inhibiting drugs on Ca21 flux were determined by direct exposure during recording as well as pretreating cells with these compounds to regulate the eicosanoid cascade. Then cells were stimulated with an agonists that normally causes an increased [Ca21]i. Agonist stimulations (MC and CPA) were performed in Ca21-free KR to measure the integrity of the intracellular stores. All drug and media changes were applied by a perfusion system which was able to exchange the entire volume of the cuvette in a matter of seconds. Background fluorescence of Fura-2-loaded cells was significant and was quantified and corrected with standard equations subtracted from fluorescence measurements [35]. All trials were performed at 22°C with an in situ experimentally determined Kd of 371 nM for astrocytoma [35] and 624 nM for meningioma cultures (unpublished) which were above published in vitro results of 135 nM [32]. Data analysis: Growth and calcium. Dose dependency of growth data were collected by manual cell count on a hemocytometer and were presented as percentage of control condition (100%) for the respective drug vehicles. Growth data were fitted to an exponential decay curve. All dose-dependent growth data points were subjected to a one-way analysis of variance (ANOVA) followed by Tukey’s multiple comparisons. Growth agonist/antagonist data and Ca21 data were analyzed quantitatively as actual cell number and Ca21 concentration estimates. These data were expressed as the mean 1/2 the standard error of mean (SEM) and were analyzed by two-
Growth: PLA2 inhibition. As depicted in Fig. 1, growth of astrocytoma cultures was inhibited in a dosedependent manner by 4-BPB with IC50 of 35 mM and quinacrine with an IC50 of 20 mM. These inhibitory effects were also produced in meningioma cultures by quinacrine with an IC50 of 2 mM and 22 mM for 4-BPB. These results suggest that PLA2 activity and/or products of this activity are necessary for basal rates of cellular proliferation for these cultures. Growth agonist/antagonist. Growth inhibition caused by PLA2 antagonism was significantly attenuated by the addition of 5 mM AA (Fig. 2). PLA2 inhibitors 4-BPB and quinacrine were added to astrocytoma and meningioma cultures at their respective IC50 as determined by dose-dependent growth curves. Dependence of the growth inhibition on the loss of the PLA2 product AA was tested by the addition of 5 mM AA to 4-BPB and quinacrine-treated cultures resulting in the reversal of antiproliferative effects. Treatment with AA alone did not cause significant changes compared to the control group; however, both 4-BPB and quinacrine groups inhibit growth significantly compared to control. Addition of AA to cultures simultaneously treated with 4-BPB or quinacrine, resulted in a significant increased cell number compared to cells treated with PLA2 inhibitors alone (P # 0.05, n 5 6). HPLC assessment of eicosanoid synthetic enzyme activity. Since administration of PLA2 inhibitors resulted in reduced proliferation of treated cultures and AA replacement restored proliferation, the possibility
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FIG. 2. Antiproliferative effects induced by PLA2 inhibition were reversed by the exogenous addition of AA. (A) astrocytoma and (B) meningioma-proliferative inhibition by PLA2 inhibitors 4-BPB and quinacrine at their respective IC50 was antagonized by the addition of the enzymatic reaction product free AA (5 mM). Data were analyzed by two-way ANOVA and demonstrated a significant antagonism of PLA2 inhibitor by the addition of AA (P # 0.05, n 5 6). Two levels of ANOVA were non-AA-pretreated cells versus AA-pretreated cells and cells without PLA2 inhibition versus those subjected to PLA2 inhibition. (a) Significant inhibition of culture proliferation induced by PLA2 blockers quinacrine and 4-BPB compared to control cultures not exposed to exogenous AA. (b) Significant preservation of proliferation in cultures treated with PLA2 inhibitors after exogenous AA pretreatment compared to those treated with inhibitors alone.
of downstream AA metabolite involvement was addressed. The activity of downstream enzymes cycloxygenase and lipoxygenase was assessed in both astrocytoma and meningioma cultures. HPLC of lipophilic extracts of cell culture homogenates preincubated with 3 H-AA substrate reveals the level of expression of functional enzymes by separating 3H products of these reactions. In vitro cytosolic incubations of both cell culture types with 3H-AA generated no reproducible detectable prostaglandins or leukotrienes. Examples of HPLC tracings for astrocytoma and meningioma are given in Fig. 3A. A smooth baseline persisted throughout the time course of the tracings except for a large peak between 59 and 61 min, which was identified by stan-
dard AA elution as unmetabolized 3H-AA. These tissue culture responses were qualitatively compared to murine spleen and feline lung (Fig. 3B), which were expected to demonstrate strong positive responses for the generation of lipoxygenase and cyclooxygenase reaction products due to immune cells and endothelium in these tissues. HPLC was performed on the lipophilic extracts of astrocytoma and meningioma cultures pretreated with phosphatidyl choline-SN2 3H-arachidonate (3H-AASN2-PC). This substrate is metabolized by PLA2 resulting in free 3H-AA. Both astrocytoma and meningioma cultures demonstrate PLA2 activity resulting in large peaks eluted from the column at 60 min on the tracings (Figs. 4 and 5). Superimposed on these matched control
FIG. 3. (A) HPLC of lipophilic extracts of cell homogenates from astrocytoma and meningioma preparations incubated with 3H-AA was performed over a 90-min elution time. Raw counts per minute (cpm) were plotted against time demonstrating a peak at 60 min for both the astrocytoma (solid line) and the meningioma (dashed line). This peak is at the appropriate elution time to be identified as unmetabolized AA when compared to the elution of control 3H-AA. (B) HPLC of lipophilic extracts of cell homogenates from feline lung and murine spleen preparations incubated with 3H-AA was performed over a 90-min elution time. Raw counts per minute (cpm) were plotted against time demonstrating many peaks eluted prior to 45 min in the cyclooxygenase solvent solubility range and lipoxygenase products are eluted after 45 min as more apolar solvent was included. Murine spleen (dashed line) and the feline lung (solid line) both demonstrated cyclooxygenase and lipoxygenase products confirming the validity of this assay.
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FIG. 4. HPLC of lipophilic extracts of cell homogenates from astrocytoma pretreated with EC50 4-BPB (A) or quinacrine (B) compared to matched control preparations incubated with 3H-AA-SN2-PC was performed over a 90-min elution time. Raw counts per minute (cpm) were plotted against time demonstrating a peak at 60 min for control preparations (dashed line) and a reduced peak at 60 min for preparations pretreated with 4-BPB or quinacrine (solid line). This peak is at the appropriate elution time to be identified as AA when compared to the elution of control 3H-AA.
tracings are HPLC tracings produced with PLA2 inhibitors 4-BPB or quinacrine demonstrating the decrease in free 3H-AA produced. Calcium. PLA2 inhibition induced a temporarily increased free cytosolic Ca21 at the expense of the integrity of the intracellular Ca21 storage, suggesting that the compromised intracellular Ca21 source may contribute to growth inhibition. All Ca21 data were obtained with the use of 4-BPB only because quinacrine was highly fluorescent and interfered with the measurement of free cytosolic [Ca21]. Intracellular changes in free [Ca21] due to PLA2 inhibition were dependent on the regulation of an intracellular source. As depicted in Fig. 6A, cytosolic Ca21 levels were elevated
rapidly after the application of 4-BPB (50 mM) in Ca21free KR solution. In order to demonstrate further the intracellular Ca21 storage involvement, cells were preincubated for 15 min with dantrolene 5 mM, an inhibitor of intracellular Ca21 store release, and subsequently exposed to 4-BPB in Ca21-free KR solution. Dantrolene antagonized the cytosolic Ca21 elevation observed in cells treated with the PLA2 inhibitor. The 4-BPB induced increase in free cytosolic [Ca21] was also antagonized by a 1-h pretreatment of cells with AA (5 mM) (Fig. 6B). This result indicates that the active role of 4-BPB in Ca21 regulation is AA dependent. More evidence that the application of 4-BPB caused a release of intracellular Ca21 stores into the cytoplas-
FIG. 5. HPLC of lipophilic extracts of cell homogenates from meningioma pretreated with EC50 4-BPB (A) or quinacrine (B) compared to matched control preparations incubated with 3H-AA-SN2-PC was performed over a 90-min elution time. Raw counts per min (cpm) were plotted against time demonstrating a peak at 60 min for control preparations (dashed line) and a reduced peak at 60 min for preparations pretreated with 4-BPB or quinacrine (solid line). This peak is at the appropriate elution time to be identified as AA when compared to the elution of control 3H-AA.
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FIG. 6. Representative tracings of free intracellular [Ca21] data using astrocytoma cells as a model in Ca21-free KR solution exemplifying the characteristic relationship between PLA2 inhibition and intracellular Ca21 stores. (A) [Ca21] in nM range is plotted as a function of time with the application of PLA2 inhibitor 4-BPB (50 mM) with and without the pretreatment of dantrolene (5 mM). Increased free cytosolic [Ca21] induced by PLA2 inhibition was blocked by preincubation for 15 min with intracellular Ca21 release inhibitor dantrolene. (B) [Ca21] in nM range is plotted as a function of time with and without a 1-h pretreatment with AA (5 mM). (C) [Ca21] in nM range is plotted as a function of time after pretreatment with 4-BPB for 20, 30, or 40 min. MC (5 mM) a muscarinic agonist was used to stimulate the release of intracellular Ca21 to assess the integrity of the cellular stores. Untreated control cells demonstrated a low resting free cytosolic [Ca21] and exhibited a [Ca21] transient increase in response to agonist stimulation. As the pretreatment of PLA2 inhibitor was applied for 20-, 30-, or 40-min intervals, the resting [Ca21] increased while the agonist-stimulated transient decreased.
mic compartment resulting in storage depletion is observed in Fig. 6C. Astrocytoma cultures were pretreated with 4-BPB for 20, 30, and 40 min; basalcytosolic-free [Ca21] levels were compared to untreated controls. The integrity of the intracellular Ca21 stores was evaluated by stimulation with agonist acetyl-bmethylcholine chloride (5 mM) (Sigma). Increased free cytosolic Ca21 induced by exposure to 4-BPB was time dependent, reaching a maximum at 40 min. The MCstimulated intracellular release decreased in magnitude as the duration of 4-BPB incubation increased and the basal-free [Ca21] measurements increased. These findings suggest that 4-BPB incubation causes intracellular Ca21 stores to release their contents into the cytosolic compartment resulting in elevated cytosolic-free [Ca21] and depleted Ca21 stores. The 4-BPB-induced free cytosolic [Ca21] increase was temporary. Figure 7 depicts the cytosolic-free [Ca21] for astrocytes exposed to 4-BPB for 24 h compared to controls under resting and MC (5 mM)-
FIG. 7. Astrocytoma resting free cytosolic [Ca21] is estimated as 135 6 29 nM in control cells and 175 6 42 nM in cells pretreated with 4-BPB (50 mM) for 24 h. Control cells were stimulated to release Ca21 from intracellular stores with 5 mM MC resulting in significant (a) free cytosolic [Ca21] increase of 380 6 178 nM. Cells pretreated with 4-BPB (50 mM) for 24 h were stimulated with 5 mM MC resulting in cytosolic Ca21 increases to 200 6 35 nM, which were significantly reduced from control stimulations (b), n 5 8 and P # 0.05.
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FIG. 8. Changes in free cytosolic [Ca21] and intracellular stores induced by PLA2 inhibition were prevented by the pretreatment of cells with AA. (A) [Ca21] nM range was quantified in astrocytoma with non-AA-treated cells (solid bars) successively measured when at rest, after treatment with 4-BPB (50 mM) for 15 min and then stimulated with MC (5 mM) in Ca21-free solution. The antagonist group pretreated with AA (5 mM) for 15 min (open bars) was successively measured at rest, after treatment with 4-BPB (50 mM) for 15 min, and then stimulated with MC (5 mM) in Ca21-free solution. (B) [Ca21] in nM range was quantified in meningioma cultures for conditions included in eight groups in Ca21-free solutions. Groups are divided into those that were not pretreated with AA (solid bars) and those that were pretreated with AA (5 mM) for 1 h (open bars). These groups were further divided into those that were at rest, 4-BPB (50 mM) pretreated (1 h), CPA (30 mM) stimulated in Ca21-free solution, and 4-BPB pretreatment for 1 h with CPA stimulation in Ca21-free solution. Data were analyzed by two-way ANOVA with P # 0.05, n 5 8. (a) PLA2 inhibition significantly increased cytosolic-free Ca21 above resting levels in non-AApretreated cells. (b) Cells pretreated with AA demonstrated a significant antagonism of the Ca21 increases induced by PLA2 inhibition. Those cells pretreated with 4-BPB alone did not demonstrate any further Ca21 increase when stimulated with MC or CPA.
stimulated conditions. Astrocytes exposed to 4-BPB for 24 h did not result in free cytosolic [Ca21] levels greater than those in control cells, suggesting that these shortterm [Ca21] increases were compensated over time. This compensation probably occurred by plasmallemmal mechanisms since internal stores were not replenished. MC (5 mM)-stimulated cells pretreated with 4-BPB for 24 h resulted in [Ca21] increases significantly less than those in MC-stimulated control cells. These results suggest that 4-BPB-induced cytosolic [Ca21] increases are transient. Cytosolic [Ca21] in PLA2-inhibited cells returns to near resting control levels over 24 h. The negative response of pretreated cells to MC stimulation indicates that the return of normal cytosolic [Ca21] was not accomplished by the endoplasmic reticulum (ER) reuptake. Calcium agonist/antagonist. Consequences of PLA2 inhibition on cytosolic [Ca21] in cultures of these neoplastic tissues are shown in Fig. 8. The short time course of cellular reactions for the astrocyte allows a single cell culture preparation to be measured for free cytosolic [Ca21] with sequential experimental treatments (Fig. 8A). However, the prolonged time course of the meningioma cellular response dictated that separate cell culture preparations be used for each group (Fig. 8B). Both culture types were exposed to 4-BPB 50 mM (astrocytoma 15 min and meningioma 1 h) resulting in elevations of free cytosolic [Ca21] significantly above the control resting level. In both cell types pretreatment with AA completely antagonized the increased [Ca21] observed from exposure to 4-BPB, but AA alone caused no changes compared to controls. To test the integrity of internal Ca21 stores, astro-
cytoma cultures were stimulated with the muscarinic agonist MC. The integrity of the meningioma internal Ca21stores was assessed by the application of 30 mM CPA. The total remaining Ca21 to be released from the ER was quantified. Neither cell type demonstrated any additional stimulated increased Ca21 measured after exposure to 4-BPB alone. These results suggest that internal stores were depleted. Both cell types demonstrated a significant increased cytosolic-free [Ca21] measured after stimulation with MC or CPA in groups rescued from 4-BPB by pretreatment with AA. These findings indicate that AA was necessary for the maintenance of internal Ca21 stores and that these stores were released to the cytosolic compartment with PLA2 inhibition. This release to the cytosol resulted in the store depletion observed. DISCUSSION
Drugs such as 4-BPB and quinacrine that inhibit the action of PLA2 produce a dose-dependent decreased growth of astrocytoma and meningioma cultures. Although the antiproliferative effect of these reagents might be due to unknown mechanisms, this possibility seems unlikely because their effects were antagonized by the addition of AA. These result agree with other literature suggesting that AA or its metabolites play a role in signal transduction and growth regulation [1–5, 15]. Most regulatory effects on proliferation are receptor-mediated and involve AA metabolites such as prostaglandins and leukotrienes. Neither the astrocytoma U373-MG nor the primary meningioma demonstrates cyclooxygenase or lipoxygenase activity necessary for
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prostaglandin or leukotriene synthesis compared to positive control tissues as determined by HPLC. These cell types do demonstrate functional PLA2 activity and are capable of generating free AA. AA itself, not its downstream metabolites, appears to be involved in proliferative control of these cell types. The role of AA in signal transduction in these cell types may be mediated by the Ca21 second messenger. This link between Ca21 and AA has been demonstrated in other cell types. Free AA modulates the intracellular Ca21 concentration in rat pancreatic acinar cells by inhibiting IP3-receptor channels and/or facilitating the plasma membrane Ca21 pump [36]. However, elevation of PLA2 activity and subsequent liberation of AA in other cell types, such as rat pituitary cells, raises the free cytosolic Ca21 level by producing downstream lipoxygenase products rather than the direct action of AA itself [37]. Intracellular Ca21 is regulated very precisely with basal-cytosolic-free Ca21 levels usually between 50 and 200 nM, while extracellular concentrations are in the millimolar range [38]. In addition, Ca21 metabolism plays a role in proliferation and differentiation. Inhibition or stimulation of cell growth mediated by Ca21 signals was revealed by indirect methods involving lowering intracellular Ca21 with chelators or increasing cytosolic Ca21 with ionophores [38]. Significant findings with Ca21 in stimulated cells were demonstrated in lymphocyte experiments inducing transformation and DNA synthesis responses with phytohemagglutinin, which were inhibited by the Ca21 chelator EGTA [39, 40]. More recently, manipulations of Ca21 metabolism demonstrated growth regulation in astrocytoma and meningioma cultures utilizing plasma membrane Ca21 channel antagonists verapamil, nifedipine, and diltiazem as well as intracellular Ca21 antagonist dantrolene [26, 41], while 24 h exposures to Ca21 ionophore ionomycin 0.3 mM proved to have cytotoxic effects (unpublished data). The importance of intracellular Ca21 stores was demonstrated by cells treated with thapsigargin which consequently did not divide [42]. Our results demonstrate that the inhibition of PLA2 causes the depletion of the intracellular Ca21 stores which may be responsible for impeding proliferation. This link among AA, Ca21 stores, and proliferation is further supported by the depletion of the intracellular Ca21 store with the irreversible inhibitor of the endoplasmic reticular Ca21 ATPase thapsigargin resulting in growth arrest reversed by the addition of AA [43]. Exogenously applied AA induced new protein synthesis of the endoplasmic reticular Ca21 ATPase and results in the recovery of the intracellular stores and growth [43]. The PLA2 antagonists 4-BPB and quinacrine demonstrated growth inhibition that was accompanied by changes in cellular Ca21 metabolism. The antagonism
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of AA production long term results in the depletion of the internal Ca21 stores. The mechanism of this depletion may be due to the loss of IP3-channel regulation, Ca21ATPase, or receptor regulation provided by AA. One alternative explanation might be that the ER Ca21 ATPase may require AA to function as an efficient reuptake mechanism. Blocking the production of free cytosolic AA may allow the ER leak mechanism to dominate. The depletion of these stores may be the cause of growth inhibition. In conclusion, inhibition of eicosanoid synthetic enzyme PLA2 leads to a decreased growth of both malignant astrocytoma and the meningioma in culture. Eicosanoid regulation has shown altered growth rates in many other cell types primarily due to prostaglandin and leukotriene receptor-mediated processes. We have demonstrated that alterations in the metabolism of AA alone greatly affect the basal-free cytosolic [Ca21] and the integrity of internal stores. Cellular Ca21 regulation has been implicated in proliferation rates of these particular cell types [26, 41]. This growth inhibitory result of PLA2 inhibition correlates well with the loss of intracellular Ca21 stores, suggesting that growth inhibition exerted by AA antagonism is mediated by the depletion of the internal Ca21 stores by facilitating a leak or impeding reuptake. REFERENCES 1.
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EX984181
PLA2 Activity Regulates Ca21 Storage-Dependent Cellular Proliferation Michael J. Petr,* Thomas C. Origitano,† and Robert D. Wurster*,†,1 *Loyola University Medical Center, Stritch School of Medicine, *Department of Neuroscience and Physiology; and †Department of Neurological Surgery, Maywood, Illinois 60153
The objective of this study is to determine the role of arachidonic acid (AA) in cell proliferation by inhibiting AA synthetic enzyme phospholipase A2 (PLA2) and to determine its involvement in the role of the second messenger intracellular calcium (Ca21). Methods used to determine the effects on proliferation of cell cultures of primary meningioma and astrocytoma U373-MG included treatment with micromolar concentrations of PLA2 inhibitors 4-bromophenacylbromide and quinacrine. Effects of these drugs on proliferation were further investigated by the application of concentrations that inhibit growth by 50% while antagonizing these agents with AA replacement. Free cytosolic Ca21 was measured with the use of fluorescent dye Fura-2 during PLA2 agonist/antagonist studies. These Ca21 measurements were performed in the absence of extracellular Ca21 to identify the contribution of intracellular Ca21 sources. PLA2 inhibition resulted in decreased growth of cultured astrocytoma and meningioma cells in a dose-dependent manner in the micromolar range. This inhibitory effect was antagonized by the addition of AA. PLA2 inhibition caused an elevation of basal-cytosolic-free [Ca21] while depleting internal Ca21 stores. These Ca21 changes were also antagonized by the addition of AA. In conclusion, these results demonstrate that AA, a PLA2 enzyme product, is involved in regulating the growth rate of these cell types. The PLA2 pathway also regulates the maintenance of the internal Ca21 stores. Ca21 is known to be a growth-related intracellular second messenger. These results suggest that the growth regulatory functions of AA are mediated by Ca21-dependent mechanisms. © 1998 Academic Press Key Words: arachidonic acid; calcium; PLA 2 ; proliferation.
INTRODUCTION
Arachidonic acid and proliferation. Eicosanoids modulate many signal transduction mechanisms. Products of 1 To whom correspondence and reprint requests should be addressed at Loyola University Medical Center, Stritch School of Medicine, Physiology, Neurological Surgery, Building 102, 2160 South 1st Ave., Maywood, IL 60153. Fax: (708) 216-6308.
0014-4827/98 $25.00 Copyright © 1998 by Academic Press All rights of reproduction in any form reserved.
arachidonic acid (AA) metabolism themselves are also directly involved in intracellular signaling [1–5]. AA pathways have been altered experimentally in other cell types to demonstrate growth-related effects. A possible relationship exists between an increased AA metabolism and some aspects of proliferative behavior [6 –14]. In endothelium and vascular smooth muscle, for example, AA pathways have been selectively manipulated resulting in altered cell growth rates [15]. Eicosanoids, arachidonic acid. Arachidonic acid is the parent bioactive lipid member of eicosanoids consisting of a C20 hydrocarbon chain with four double bonds and can be both membrane bound and cytosolic. Spatially, eicosanoids freely cross cell membranes and act in an autocrine/paracrine capacity [16]. Temporally, stimulus-induced AA production occurs after short-term second messengers, such as inositol phosphates, but precedes the occurrence of long-term signal transduction events such as protein phosphorylations [17]. The eicosanoid cascade may be induced by Ca21dependent pathways (phosphatidylcholine substrate) and Ca21-independent pathways (phosphatidylethanolamine substrate) and may be mediated by phospholipase C (PLC)-inositol trisphosphate (IP3)-Ca21 or protein kinase C (PKC) activation of phospholipase A2 (PLA2) [18 –20]. Receptors may also be directly coupled to PLA2-associated G-proteins [21]. PLA2 in astrocytes may be activated by ATP via purinergic receptors linked to G-proteins [22]. In addition to the activation of PLA2, AA is produced by alternative pathways. PLC or phospholipase D (PLD) activation generates accessible diacylglycerol (DAG), a substrate for DAG-lipase or monoglycerol lipase resulting in the generation of AA [23]. Once cleaved from the membrane by PLA2 or other pathways, AA may be metabolized by cyclooxygenase and lipoxygenase enzymes to generate prostaglandins (PG) and leukotrienes (LT). These eicosanoid products may result in a variety of receptor-mediated effects. Understanding the eicosanoid-related cellular mechanisms offers promising insights into regulation of cellular proliferation. The major focus of this paper is eicosanoid-mediated mechanisms affecting regulation of proliferation. These eicosanoid-mediated effects are suspected to occur due to the link of these lipid
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species to alterations in calcium (Ca21)-mediated signal transduction. MATERIALS AND METHODS Cell culture, cell line characteristics of U-373-MG human astrocytoma. These cultured cells were the product of a primary tumor originating from a 61-year-old, blood type A1 caucasian male by B. Westmark [24] and was purchased from American Type Culture Collection (Rockville, MD). These cells were tumorigenic in nude mice forming grade III astrocytomas and producing glial fibrillary acidic protein (GFAP) [25]. The growth rate of these cells was effected by alteration of calcium (Ca21) metabolism [26]. Cell culture, cell line characteristics of primary meningioma. Fresh tissue was retrieved from the surgical field. Tissue was aseptically placed in Earl’s balanced salt solution (EBSS) with penicillin 10 K units/L, streptomycin 10 mg/L, and amphotericin B 25 mg/L (Sigma St Louis, MO). This tissue was mechanically reduced to 2 to 5-mm3 pieces in a laminar flow hood. Tumor particles were incubated for 24 h at 37°C in 20 ml of minimal essential medium (MEM) with 10% fetal bovine serum (FBS)(1). Tumor cell isolation was achieved with MEM1 supplemented with 1 mg/ml collagenase A (Boehringer Mannheim, Indianapolis, IN), and 100 ml of 1 mg/ml deoxyribonuclease (Sigma). After enzymatic digestion the tissue was forced through 60 (0.23 mm) and 150 (0.104 mm) steel mesh (Sigma). Suspended tissue and fluid were split into two 150-ml flasks (Falcon, England). Meningioma culture use was limited to the first 5 passages due to unreliable characteristics that may occur with later passages. Meningioma cultures maintain tumor characteristics in culture as determined by light and electron microscopy and immunohistochemical studies [27]. Each experiment was performed with as many as three different tumors as culture sources. No effort was made to specifically select for age, gender, or tumor location. All tumors and cultures were characterized by histopathology and immunocytochemistry as meningioma ranging from benign to anaplastic. Culture maintenance and growth. Cultures were grown in a humidified incubator at 37°C with gas concentrations of 5% CO2/95% air, and media was replaced every 3 or 4 days. Cells were harvested for experimentation by adding 0.25% trypsin (Sigma) and 50 mM ethylene glycol-bis(b-aminoethylether) N,N,N9,N9,-tetraacetic acid (EGTA) for 8 min. After trypsin and EGTA, effects were quenched by the addition of serum and dilution. Cells were separated from solution by centrifugation. Cells from confluent cultures were harvested for growth studies and reseeded in 35 3 10-mm culture dishes (Fisher Scientific, Pittsburgh, PA) at a density of 2.5 3 105 cells/dish for the astrocytoma and 1.5 3 105 cells/dish for the meningioma. Pharmacological treatment for growth. Stock solutions of 10 mM 4-bromophenacylbromide (4-BPB) (Sigma) were prepared in 70% ethanol and were diluted to final concentrations in 2-ml petri culture dishes. Final ethanol concentrations in cultures were less than 0.35%. Studies show no difference in growth using 0.35% ethanol verses water controls (data not shown). Quinacrine (Sigma) was prepared in a stock solution of 10 mM in double-distilled water. All stock solutions were stored at 4°C and protected from exposure to light. Concentrations ranging above and below the EC50 for each drug and cell type were tested for effects on growth. Reagent administration was initiated 24 h postseeding. Reagents and media were changed daily for 3 days, and growth measurements were made on Day 4. Cells were harvested by trypsinization and counted with a hemocytometer using trypan blue exclusion to determine a change in growth rate as compared to matched control independent of cytotoxicity. Growth inhibition by PLA2 inhibitors quinacrine and 4-BPB at half their maximal effective dose was antagonized with the addition of 5 mM AA. AA and the PLA2 inhibitors were replaced with serum-
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containing media twice daily for 3 days. Cells were then harvested and counted on Day 4. High performance liquid chromatography (HPLC) methods. Cells from four confluent 75-ml culture flasks were harvested by scraping. These cells were spun to a pellet and washed with 5 ml of Dulbecco’s buffer containing (in mM): 2.7 KCl, 1.5 KH2PO4, 137 NaCl, 8.1 NaH2PO4 z H2O, 2 glucose, and 1 CaCl2 at pH 7.4. Preparations were sonicated to rupture cells for a protocol similar to that used by Voltera et al. [28]. Sonicated cells were incubated with 1 mCi tritiated arachidonic acid (3H-AA) (210 Ci/mmol, 7770.0 GBq/mmol,) or 1 mCi tritiated phosphatidylcholine, L-a-1-stearoyl-2-arachidonyl [arachidonyl-5,6,8,9,11,12,14,15-3H (N)] (160.0 Ci/mmol, 5920.0 GBq/mmol) (3H-PC) (Dupont, NEN) at 37°C. The assay was terminated by the addition of 265 ml of 5 N formic acid and cooling on ice. Preparations were separated three times with ethyl acetate in a 1 to 1-vol ratio. The organic phase was then evaporated in a speed evacuation centrifuge. Dry lipid was redisolved in mobile phase B and loaded into the autoinjector. The aqueous phase was saved for protein analysis. Reverse-phase HPLC was performed by the method of Powell [29]. A gradient of polar mobile phase A to nonpolar mobile phase B was used with pumps and gradient mixer (Rainin). The gradient at 0 min was 100% mobile phase A (75% H 2 O, 25% CH 3 CN, 0.001% CF3COOH), 40 min mobile phase A was 20%, while mobile phase B was 80%, 60 –90 min mobile phase B was 100% (7.5% H2O, 54% CH3OH, 38.5% CH3CN, 0.001% CF3COOH). An autoinjector (Shamadzu), ultraviolet detector (Waters), and Flo-one/b scintillation radiodetector (Canberra-Packard) were connected in series. Cell numbers used for each experiment were standardized by normalization to protein. The ultraviolet detector was used to standardize elution times of a number of nonradioactive leukotriene samples such as LtB4, LtC4, 5HETE and 12HETE. Measurement of free intracellular calcium concentrations: Solutions and loading Fura-2-AM. Fluorescent Ca21 indicator Fura-2acetoxymethyl ester (Molecular Probes, Eugene, OR) was chosen for this study based on the published dissociation constant (Kd) (224 nM at 37°C and 135 nM at 22°C). These Kd values theoretically allowed accurate measurements of the free cytosolic Ca21 from resting values of 10 nM to the mM range. Fura-2-AM is lipophilic and not soluble in aqueous solutions, but does readily cross the lipid bilayer. Once the AM-ester crosses into the cytosol, nonspecific esterases cleave the ester, resulting in a charged free acid form which is not lost from the cell as rapidly as the parent lipid soluble compound [30]. Since AM-esters are insoluble in aqueous solutions, indicator was placed in micelles of the dispersing agent pluronic acid (Molecular Probes). Fura-2-AM was dissolved in dimethylsulfoxide (DMSO) (Sigma) at a concentration of 1 mg/ml for stock solution. The indicator preparation was added to Krebs-bovine serum albumin (BSA) 1 mg/ml solution resulting in an indicator end concentration of 1 mM [31]. Cell cultures were grown as a monolayer on polylysine-coated glass coverslips. These coverslips were placed in a 35 3 10-mm culture dish (Becton Dickenson, Lincoln Park, NJ). All serum was removed by washing with Krebs Ringer solution (KR). Coverslips containing cultured cells were covered with 1 ml of loading solution and were allowed to incubate for 1 h. Coverslips were then rinsed twice with KR and were ready for use. To prevent photo bleaching, all preparations with fluorescent indicators were used under conditions that provide protection from light [30]. Krebs Ringer solution contained: 125 mM NaCl, 1.2 mM KH2PO4, 1.2 mM MgSO4, 5 mM NaHCO3, 25 mM Hepes, 6 mM glucose (Sigma), 5 mM KCl, and 1.3 mM CaCl2 (Mallinckrodt, Paris , KE). Ca21- free KR was made as suggested above with the omission of CaCl2. The pH was adjusted to 7.4 with 1 N NaOH. Calcium measurement. Glass coverslips with the monolayer were placed in a cuvette with a perfusion apparatus. This cuvette was placed in the spectrophotometer chamber (Perkin Elmer, Buckinghamshire, England). Excitation wavelengths of 340 and 380 nm were used for Fura-2 bound and unbound to Ca21. Emission measure-
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FIG. 1. Dose dependent growth data are expressed as the mean and standard deviation of percentage of control. Solid squares (astrocytoma) and solid triangles (meningioma) demonstrate a dose-dependent inhibition of growth compared to untreated controls. (A) PLA2 inhibitor 4-BPB demonstrated an IC50 of 35 and 22 mM for astrocytoma and meningioma, respectively. (B) PLA2 inhibitor quinacrine demonstrated an IC50 of 6 and 2 mM for astrocytoma and meningioma, respectively. Growth curves are analyzed by one-way ANOVA and found to be statistically significant with P # 0.05, n 5 8.
ments in the 510 nm range were taken during fast frequency changes between excitation filters 340 and 380 nm to produce data points with comparable temporal relationships. Free-Ca21 concentrations were calculated with the Grynkiewicz equation [32].
way ANOVA and multiple comparisons. Data which have a P value # 0.05 were considered to be statistically significant.
RESULTS Free [Ca21]I nM 5 k d[(R 2 R min /R max 2 R)(b)]. Fluorescence scanning time was between 300 and 600 s which was enough time to measure intracellular changes due to positive controls such as acetylmethylcholine (MC) (5 mM) or cyclopiazonic acid (CPA) (30 mM). MC is a muscarinic-specific agonist which activates phospholipase C and inositol trisphosphate-induced intracellular Ca21 release from the endoplasmic reticulum [33]. CPA is a reversible inhibitor of the endoplasmic reticular Ca21ATPase [34]. Measurements were determined for control and experimental groups in order to determine the result of drug application on basal intracellular Ca21 ([Ca21]i) as well as agonist-stimulated cells. Effects of eicosanoid-inhibiting drugs on Ca21 flux were determined by direct exposure during recording as well as pretreating cells with these compounds to regulate the eicosanoid cascade. Then cells were stimulated with an agonists that normally causes an increased [Ca21]i. Agonist stimulations (MC and CPA) were performed in Ca21-free KR to measure the integrity of the intracellular stores. All drug and media changes were applied by a perfusion system which was able to exchange the entire volume of the cuvette in a matter of seconds. Background fluorescence of Fura-2-loaded cells was significant and was quantified and corrected with standard equations subtracted from fluorescence measurements [35]. All trials were performed at 22°C with an in situ experimentally determined Kd of 371 nM for astrocytoma [35] and 624 nM for meningioma cultures (unpublished) which were above published in vitro results of 135 nM [32]. Data analysis: Growth and calcium. Dose dependency of growth data were collected by manual cell count on a hemocytometer and were presented as percentage of control condition (100%) for the respective drug vehicles. Growth data were fitted to an exponential decay curve. All dose-dependent growth data points were subjected to a one-way analysis of variance (ANOVA) followed by Tukey’s multiple comparisons. Growth agonist/antagonist data and Ca21 data were analyzed quantitatively as actual cell number and Ca21 concentration estimates. These data were expressed as the mean 1/2 the standard error of mean (SEM) and were analyzed by two-
Growth: PLA2 inhibition. As depicted in Fig. 1, growth of astrocytoma cultures was inhibited in a dosedependent manner by 4-BPB with IC50 of 35 mM and quinacrine with an IC50 of 20 mM. These inhibitory effects were also produced in meningioma cultures by quinacrine with an IC50 of 2 mM and 22 mM for 4-BPB. These results suggest that PLA2 activity and/or products of this activity are necessary for basal rates of cellular proliferation for these cultures. Growth agonist/antagonist. Growth inhibition caused by PLA2 antagonism was significantly attenuated by the addition of 5 mM AA (Fig. 2). PLA2 inhibitors 4-BPB and quinacrine were added to astrocytoma and meningioma cultures at their respective IC50 as determined by dose-dependent growth curves. Dependence of the growth inhibition on the loss of the PLA2 product AA was tested by the addition of 5 mM AA to 4-BPB and quinacrine-treated cultures resulting in the reversal of antiproliferative effects. Treatment with AA alone did not cause significant changes compared to the control group; however, both 4-BPB and quinacrine groups inhibit growth significantly compared to control. Addition of AA to cultures simultaneously treated with 4-BPB or quinacrine, resulted in a significant increased cell number compared to cells treated with PLA2 inhibitors alone (P # 0.05, n 5 6). HPLC assessment of eicosanoid synthetic enzyme activity. Since administration of PLA2 inhibitors resulted in reduced proliferation of treated cultures and AA replacement restored proliferation, the possibility
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FIG. 2. Antiproliferative effects induced by PLA2 inhibition were reversed by the exogenous addition of AA. (A) astrocytoma and (B) meningioma-proliferative inhibition by PLA2 inhibitors 4-BPB and quinacrine at their respective IC50 was antagonized by the addition of the enzymatic reaction product free AA (5 mM). Data were analyzed by two-way ANOVA and demonstrated a significant antagonism of PLA2 inhibitor by the addition of AA (P # 0.05, n 5 6). Two levels of ANOVA were non-AA-pretreated cells versus AA-pretreated cells and cells without PLA2 inhibition versus those subjected to PLA2 inhibition. (a) Significant inhibition of culture proliferation induced by PLA2 blockers quinacrine and 4-BPB compared to control cultures not exposed to exogenous AA. (b) Significant preservation of proliferation in cultures treated with PLA2 inhibitors after exogenous AA pretreatment compared to those treated with inhibitors alone.
of downstream AA metabolite involvement was addressed. The activity of downstream enzymes cycloxygenase and lipoxygenase was assessed in both astrocytoma and meningioma cultures. HPLC of lipophilic extracts of cell culture homogenates preincubated with 3 H-AA substrate reveals the level of expression of functional enzymes by separating 3H products of these reactions. In vitro cytosolic incubations of both cell culture types with 3H-AA generated no reproducible detectable prostaglandins or leukotrienes. Examples of HPLC tracings for astrocytoma and meningioma are given in Fig. 3A. A smooth baseline persisted throughout the time course of the tracings except for a large peak between 59 and 61 min, which was identified by stan-
dard AA elution as unmetabolized 3H-AA. These tissue culture responses were qualitatively compared to murine spleen and feline lung (Fig. 3B), which were expected to demonstrate strong positive responses for the generation of lipoxygenase and cyclooxygenase reaction products due to immune cells and endothelium in these tissues. HPLC was performed on the lipophilic extracts of astrocytoma and meningioma cultures pretreated with phosphatidyl choline-SN2 3H-arachidonate (3H-AASN2-PC). This substrate is metabolized by PLA2 resulting in free 3H-AA. Both astrocytoma and meningioma cultures demonstrate PLA2 activity resulting in large peaks eluted from the column at 60 min on the tracings (Figs. 4 and 5). Superimposed on these matched control
FIG. 3. (A) HPLC of lipophilic extracts of cell homogenates from astrocytoma and meningioma preparations incubated with 3H-AA was performed over a 90-min elution time. Raw counts per minute (cpm) were plotted against time demonstrating a peak at 60 min for both the astrocytoma (solid line) and the meningioma (dashed line). This peak is at the appropriate elution time to be identified as unmetabolized AA when compared to the elution of control 3H-AA. (B) HPLC of lipophilic extracts of cell homogenates from feline lung and murine spleen preparations incubated with 3H-AA was performed over a 90-min elution time. Raw counts per minute (cpm) were plotted against time demonstrating many peaks eluted prior to 45 min in the cyclooxygenase solvent solubility range and lipoxygenase products are eluted after 45 min as more apolar solvent was included. Murine spleen (dashed line) and the feline lung (solid line) both demonstrated cyclooxygenase and lipoxygenase products confirming the validity of this assay.
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FIG. 4. HPLC of lipophilic extracts of cell homogenates from astrocytoma pretreated with EC50 4-BPB (A) or quinacrine (B) compared to matched control preparations incubated with 3H-AA-SN2-PC was performed over a 90-min elution time. Raw counts per minute (cpm) were plotted against time demonstrating a peak at 60 min for control preparations (dashed line) and a reduced peak at 60 min for preparations pretreated with 4-BPB or quinacrine (solid line). This peak is at the appropriate elution time to be identified as AA when compared to the elution of control 3H-AA.
tracings are HPLC tracings produced with PLA2 inhibitors 4-BPB or quinacrine demonstrating the decrease in free 3H-AA produced. Calcium. PLA2 inhibition induced a temporarily increased free cytosolic Ca21 at the expense of the integrity of the intracellular Ca21 storage, suggesting that the compromised intracellular Ca21 source may contribute to growth inhibition. All Ca21 data were obtained with the use of 4-BPB only because quinacrine was highly fluorescent and interfered with the measurement of free cytosolic [Ca21]. Intracellular changes in free [Ca21] due to PLA2 inhibition were dependent on the regulation of an intracellular source. As depicted in Fig. 6A, cytosolic Ca21 levels were elevated
rapidly after the application of 4-BPB (50 mM) in Ca21free KR solution. In order to demonstrate further the intracellular Ca21 storage involvement, cells were preincubated for 15 min with dantrolene 5 mM, an inhibitor of intracellular Ca21 store release, and subsequently exposed to 4-BPB in Ca21-free KR solution. Dantrolene antagonized the cytosolic Ca21 elevation observed in cells treated with the PLA2 inhibitor. The 4-BPB induced increase in free cytosolic [Ca21] was also antagonized by a 1-h pretreatment of cells with AA (5 mM) (Fig. 6B). This result indicates that the active role of 4-BPB in Ca21 regulation is AA dependent. More evidence that the application of 4-BPB caused a release of intracellular Ca21 stores into the cytoplas-
FIG. 5. HPLC of lipophilic extracts of cell homogenates from meningioma pretreated with EC50 4-BPB (A) or quinacrine (B) compared to matched control preparations incubated with 3H-AA-SN2-PC was performed over a 90-min elution time. Raw counts per min (cpm) were plotted against time demonstrating a peak at 60 min for control preparations (dashed line) and a reduced peak at 60 min for preparations pretreated with 4-BPB or quinacrine (solid line). This peak is at the appropriate elution time to be identified as AA when compared to the elution of control 3H-AA.
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FIG. 6. Representative tracings of free intracellular [Ca21] data using astrocytoma cells as a model in Ca21-free KR solution exemplifying the characteristic relationship between PLA2 inhibition and intracellular Ca21 stores. (A) [Ca21] in nM range is plotted as a function of time with the application of PLA2 inhibitor 4-BPB (50 mM) with and without the pretreatment of dantrolene (5 mM). Increased free cytosolic [Ca21] induced by PLA2 inhibition was blocked by preincubation for 15 min with intracellular Ca21 release inhibitor dantrolene. (B) [Ca21] in nM range is plotted as a function of time with and without a 1-h pretreatment with AA (5 mM). (C) [Ca21] in nM range is plotted as a function of time after pretreatment with 4-BPB for 20, 30, or 40 min. MC (5 mM) a muscarinic agonist was used to stimulate the release of intracellular Ca21 to assess the integrity of the cellular stores. Untreated control cells demonstrated a low resting free cytosolic [Ca21] and exhibited a [Ca21] transient increase in response to agonist stimulation. As the pretreatment of PLA2 inhibitor was applied for 20-, 30-, or 40-min intervals, the resting [Ca21] increased while the agonist-stimulated transient decreased.
mic compartment resulting in storage depletion is observed in Fig. 6C. Astrocytoma cultures were pretreated with 4-BPB for 20, 30, and 40 min; basalcytosolic-free [Ca21] levels were compared to untreated controls. The integrity of the intracellular Ca21 stores was evaluated by stimulation with agonist acetyl-bmethylcholine chloride (5 mM) (Sigma). Increased free cytosolic Ca21 induced by exposure to 4-BPB was time dependent, reaching a maximum at 40 min. The MCstimulated intracellular release decreased in magnitude as the duration of 4-BPB incubation increased and the basal-free [Ca21] measurements increased. These findings suggest that 4-BPB incubation causes intracellular Ca21 stores to release their contents into the cytosolic compartment resulting in elevated cytosolic-free [Ca21] and depleted Ca21 stores. The 4-BPB-induced free cytosolic [Ca21] increase was temporary. Figure 7 depicts the cytosolic-free [Ca21] for astrocytes exposed to 4-BPB for 24 h compared to controls under resting and MC (5 mM)-
FIG. 7. Astrocytoma resting free cytosolic [Ca21] is estimated as 135 6 29 nM in control cells and 175 6 42 nM in cells pretreated with 4-BPB (50 mM) for 24 h. Control cells were stimulated to release Ca21 from intracellular stores with 5 mM MC resulting in significant (a) free cytosolic [Ca21] increase of 380 6 178 nM. Cells pretreated with 4-BPB (50 mM) for 24 h were stimulated with 5 mM MC resulting in cytosolic Ca21 increases to 200 6 35 nM, which were significantly reduced from control stimulations (b), n 5 8 and P # 0.05.
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FIG. 8. Changes in free cytosolic [Ca21] and intracellular stores induced by PLA2 inhibition were prevented by the pretreatment of cells with AA. (A) [Ca21] nM range was quantified in astrocytoma with non-AA-treated cells (solid bars) successively measured when at rest, after treatment with 4-BPB (50 mM) for 15 min and then stimulated with MC (5 mM) in Ca21-free solution. The antagonist group pretreated with AA (5 mM) for 15 min (open bars) was successively measured at rest, after treatment with 4-BPB (50 mM) for 15 min, and then stimulated with MC (5 mM) in Ca21-free solution. (B) [Ca21] in nM range was quantified in meningioma cultures for conditions included in eight groups in Ca21-free solutions. Groups are divided into those that were not pretreated with AA (solid bars) and those that were pretreated with AA (5 mM) for 1 h (open bars). These groups were further divided into those that were at rest, 4-BPB (50 mM) pretreated (1 h), CPA (30 mM) stimulated in Ca21-free solution, and 4-BPB pretreatment for 1 h with CPA stimulation in Ca21-free solution. Data were analyzed by two-way ANOVA with P # 0.05, n 5 8. (a) PLA2 inhibition significantly increased cytosolic-free Ca21 above resting levels in non-AApretreated cells. (b) Cells pretreated with AA demonstrated a significant antagonism of the Ca21 increases induced by PLA2 inhibition. Those cells pretreated with 4-BPB alone did not demonstrate any further Ca21 increase when stimulated with MC or CPA.
stimulated conditions. Astrocytes exposed to 4-BPB for 24 h did not result in free cytosolic [Ca21] levels greater than those in control cells, suggesting that these shortterm [Ca21] increases were compensated over time. This compensation probably occurred by plasmallemmal mechanisms since internal stores were not replenished. MC (5 mM)-stimulated cells pretreated with 4-BPB for 24 h resulted in [Ca21] increases significantly less than those in MC-stimulated control cells. These results suggest that 4-BPB-induced cytosolic [Ca21] increases are transient. Cytosolic [Ca21] in PLA2-inhibited cells returns to near resting control levels over 24 h. The negative response of pretreated cells to MC stimulation indicates that the return of normal cytosolic [Ca21] was not accomplished by the endoplasmic reticulum (ER) reuptake. Calcium agonist/antagonist. Consequences of PLA2 inhibition on cytosolic [Ca21] in cultures of these neoplastic tissues are shown in Fig. 8. The short time course of cellular reactions for the astrocyte allows a single cell culture preparation to be measured for free cytosolic [Ca21] with sequential experimental treatments (Fig. 8A). However, the prolonged time course of the meningioma cellular response dictated that separate cell culture preparations be used for each group (Fig. 8B). Both culture types were exposed to 4-BPB 50 mM (astrocytoma 15 min and meningioma 1 h) resulting in elevations of free cytosolic [Ca21] significantly above the control resting level. In both cell types pretreatment with AA completely antagonized the increased [Ca21] observed from exposure to 4-BPB, but AA alone caused no changes compared to controls. To test the integrity of internal Ca21 stores, astro-
cytoma cultures were stimulated with the muscarinic agonist MC. The integrity of the meningioma internal Ca21stores was assessed by the application of 30 mM CPA. The total remaining Ca21 to be released from the ER was quantified. Neither cell type demonstrated any additional stimulated increased Ca21 measured after exposure to 4-BPB alone. These results suggest that internal stores were depleted. Both cell types demonstrated a significant increased cytosolic-free [Ca21] measured after stimulation with MC or CPA in groups rescued from 4-BPB by pretreatment with AA. These findings indicate that AA was necessary for the maintenance of internal Ca21 stores and that these stores were released to the cytosolic compartment with PLA2 inhibition. This release to the cytosol resulted in the store depletion observed. DISCUSSION
Drugs such as 4-BPB and quinacrine that inhibit the action of PLA2 produce a dose-dependent decreased growth of astrocytoma and meningioma cultures. Although the antiproliferative effect of these reagents might be due to unknown mechanisms, this possibility seems unlikely because their effects were antagonized by the addition of AA. These result agree with other literature suggesting that AA or its metabolites play a role in signal transduction and growth regulation [1–5, 15]. Most regulatory effects on proliferation are receptor-mediated and involve AA metabolites such as prostaglandins and leukotrienes. Neither the astrocytoma U373-MG nor the primary meningioma demonstrates cyclooxygenase or lipoxygenase activity necessary for
PROLIFERATION DEPENDENCE ON ARACHIDONATE
prostaglandin or leukotriene synthesis compared to positive control tissues as determined by HPLC. These cell types do demonstrate functional PLA2 activity and are capable of generating free AA. AA itself, not its downstream metabolites, appears to be involved in proliferative control of these cell types. The role of AA in signal transduction in these cell types may be mediated by the Ca21 second messenger. This link between Ca21 and AA has been demonstrated in other cell types. Free AA modulates the intracellular Ca21 concentration in rat pancreatic acinar cells by inhibiting IP3-receptor channels and/or facilitating the plasma membrane Ca21 pump [36]. However, elevation of PLA2 activity and subsequent liberation of AA in other cell types, such as rat pituitary cells, raises the free cytosolic Ca21 level by producing downstream lipoxygenase products rather than the direct action of AA itself [37]. Intracellular Ca21 is regulated very precisely with basal-cytosolic-free Ca21 levels usually between 50 and 200 nM, while extracellular concentrations are in the millimolar range [38]. In addition, Ca21 metabolism plays a role in proliferation and differentiation. Inhibition or stimulation of cell growth mediated by Ca21 signals was revealed by indirect methods involving lowering intracellular Ca21 with chelators or increasing cytosolic Ca21 with ionophores [38]. Significant findings with Ca21 in stimulated cells were demonstrated in lymphocyte experiments inducing transformation and DNA synthesis responses with phytohemagglutinin, which were inhibited by the Ca21 chelator EGTA [39, 40]. More recently, manipulations of Ca21 metabolism demonstrated growth regulation in astrocytoma and meningioma cultures utilizing plasma membrane Ca21 channel antagonists verapamil, nifedipine, and diltiazem as well as intracellular Ca21 antagonist dantrolene [26, 41], while 24 h exposures to Ca21 ionophore ionomycin 0.3 mM proved to have cytotoxic effects (unpublished data). The importance of intracellular Ca21 stores was demonstrated by cells treated with thapsigargin which consequently did not divide [42]. Our results demonstrate that the inhibition of PLA2 causes the depletion of the intracellular Ca21 stores which may be responsible for impeding proliferation. This link among AA, Ca21 stores, and proliferation is further supported by the depletion of the intracellular Ca21 store with the irreversible inhibitor of the endoplasmic reticular Ca21 ATPase thapsigargin resulting in growth arrest reversed by the addition of AA [43]. Exogenously applied AA induced new protein synthesis of the endoplasmic reticular Ca21 ATPase and results in the recovery of the intracellular stores and growth [43]. The PLA2 antagonists 4-BPB and quinacrine demonstrated growth inhibition that was accompanied by changes in cellular Ca21 metabolism. The antagonism
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of AA production long term results in the depletion of the internal Ca21 stores. The mechanism of this depletion may be due to the loss of IP3-channel regulation, Ca21ATPase, or receptor regulation provided by AA. One alternative explanation might be that the ER Ca21 ATPase may require AA to function as an efficient reuptake mechanism. Blocking the production of free cytosolic AA may allow the ER leak mechanism to dominate. The depletion of these stores may be the cause of growth inhibition. In conclusion, inhibition of eicosanoid synthetic enzyme PLA2 leads to a decreased growth of both malignant astrocytoma and the meningioma in culture. Eicosanoid regulation has shown altered growth rates in many other cell types primarily due to prostaglandin and leukotriene receptor-mediated processes. We have demonstrated that alterations in the metabolism of AA alone greatly affect the basal-free cytosolic [Ca21] and the integrity of internal stores. Cellular Ca21 regulation has been implicated in proliferation rates of these particular cell types [26, 41]. This growth inhibitory result of PLA2 inhibition correlates well with the loss of intracellular Ca21 stores, suggesting that growth inhibition exerted by AA antagonism is mediated by the depletion of the internal Ca21 stores by facilitating a leak or impeding reuptake. REFERENCES 1.
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