ARCHIVES
OF BIOCHEMISTRY
AND
BIOPHYSICS
Vol. 286, No. 1, April, pp. 293-299, 1991
Localization and Characterization of Phospholipase in Mouse Mammary Gland-Derived Cells Marion
A,
R. Steiner
Department of Microbiology and Immunology, Medical Center, Room MS 413, University of Kentucky, Lexington, Kentucky 40536
Received August 27, 1990, and in revised form December 6, 1990
Phospholipase A2 (PLA2) can participate in the regulation of eicosanoid biosynthesis via PLAz-mediated control of the release of arachidonic acid from phospholipids. Arachidonoyl-hydrolyzing PLA,s were examined in cells from normal mouse mammary glands and mammary carcinomas. Tumor-derived cells exhibited significant PLAz activity(ies) with arachidonoyl containing phosphatidylcholine and phosphatidylethanolamine as substrates in cell-free assays. In contrast, arachidonoyl containing phosphatidylinositol was a poor substrate. When phosphatidylcholines with varying sn-2 fatty acyl groups were tested as substrates, activity was highest with the arachidonoyl containing lipid. The pH profiles for hydrolysis of phosphatidylcholine and phosphatidylethanolamine differed; all other aspects of PLA,-mediated hydrolysis of these two substrates were similar including a Ca2+ requirement for activity. Moreover, Ca2+ affected the subcellular localization of the enzyme activity. Activity was predominately in the supernatant fraction when cells were harvested in an EGTA (ethylene glycol bis(@-aminoethylether)-N,N,N’,N’-tetraacetic acid) containing buffer and largely in the particulate fraction when cells were harvested in a buffer containing free Ca2+. The localization of activity could be modulated from the supernatant fraction to the particulate fraction by recentrifugation in the presence of Ca2+. Normal gland-derived cells contained a PLAz activity with properties similar to those of the tumor-derived cells. There was a significant difference in the level of activity in the normal versus tumor cells, the normal gland-derived cells had less than half the PLAz activity of the carcinomaderived cells. o lee1 Academic PPXSS, IN.
The arachidonic acid metabolites, prostaglandins, have been implicated in the development of primary mammary carcinomas and in metastasis of mammary neoplasias (l8). In addition, arachidonic acid metabolites appear to be important in normal gland function since eicosanoids 0003-9861/91$3.00 Copyright 0 1991 by Academic Press, All rights of reproduction in any form
stimulate normal cell proliferation (9). Regulation of eicosanoid synthesis is mediated in part via control of the release of arachidonic acid from glycerolipids (10). Phospholipase A2 (PLAs)’ has been implicated in the regulation of eicosanoid synthesis via its involvement in the release of arachidonic acid from phospholipids (10). For example, increased PLAs activity has been associated with increased eicosanoid synthesis in f-Met-Leu-Phe-stimulated macrophages and interleukin l-treated fibroblasts and chondrocytes (11-13). The status of PLAz activity in neoplasia has been examined in only a limited number of studies. Cell-free preparations from rat mammary carcinomas and gastric carcinomatous mucosa exhibited an increased PLAs activity as compared to preparations from normal glands and normal mucosa, respectively (14, 15). In addition, PLAs activity was higher in cultured rat fibroblasts transfected with the ras oncogene than in nontransfected control cells (16, 17). Specificity of activity with respect to the fatty acyl group in the sn-2 position and PLA2 properties were not addressed in these studies. PLA,s participate in a range of functions including phospholipid degradation, phospholipid remodeling, and regulation of free arachidonic acid levels (reviewed in (1820)), hence PLAss involved in different functions are likely to have different characteristics. With respect to specificity for an arachidonoyl group in the sn-2 position, three classes of PLAss have been observed: (i) those exhibiting a specificity for arachidonoyl groups (21-26), (ii) those exhibiting no fatty acyl specificity (27-30), and (iii) those which preferentially hydrolyze phospholipids containing fatty acyl groups other than an arachidonoyl group (31, 32). The present study was undertaken to ascertain if mouse mammary gland-derived cells have a PLA, activ1 Abbreviations used: BSA, bovine serum albumin; Chaps, 3-[(3-cholamidopropyl)-dimethylammonio]-l-propanesulfonate; DTT, dithiothreitol; EGTA, ethylene glycol his@-aminoethylether)-N,N,N:N’-tetraacetic acid; PBS, magnesium and calcium free phosphate-buffered saline; PLA, phospholipase A; TLC, thin layer chromatography. 293
Inc. reserved.
294
MARION
R. STEINER
ity(ies) which preferentially hydrolyzes phospholipids containing arachidonoyl residues, to characterize this activity, and to determine the status of this PLAz activity in mammary carcinoma-derived cells as compared to normal gland-derived cells. MATERIALS
AND
METHODS
I-Stearoyl-2[3H] arachidonoyl-sn-glycero-3-phosphoMaterids. choline (91 Ci/mmol), l-palmitoyl-2-[‘“Clarachidonoyl-sn-glycero-3phosphoethanolamine (52 mCi/mmol), l-stearoyl-2-[“Clarachidonoylsn-glycero-3-phosphoinositol (30 mCi/mmol), 1-palmitoyl-2-[sH] palmitoyl-sn-glycero-3-phosphocholine (57 Ci/mmol), 1-palmitoyl-2[“C]linoleoyl-sn-glycero-3-phosphocholine (58 mCi/mmol), and l-palmitoyl-2-[1”C]oleoyl-sn-glycero-3-phosphocholine (53 mCi/mmol) were obtained from DuPont-NEN Research Products (Boston, MA). The source of l-stearoyl-2-arachidonoyl-sn-glycero-3-phosphocholine was Avanti Polar Lipids (Birmingham, AL); Hepes, dithiothreitol (DTT), and leupeptin were from Calbiochem (San Diego, CA); all other biochemicals used in the PLAx assay were from Sigma Chemical Co. (St. Louis, MO). Bovine pituitary extract and Mite+ serum extender were obtained from Collaborative Research, Inc (Bedford, MA); other tissue culture reagents were obtained from GIBCO Laboratories (Grand Island, NY) and Sigma Chemical Co. Thin layer chromatography (TLC) plates were from Analtech (Newark, DE); Beckman Biosolv-3 was from Beckman Instruments (Irvine, CA). Cells and cell culture. The MTV-L/BALB Cl 2 and DMBA-P/BALB Cl 4 cell lines were derived from mouse mammary carcinomas which had viral and chemical carcinogen etiologies respectively (33, 34). The COMMA-D cell line (generously provided by Dr. D. Medina, Baylor College of Medicine, Houston, TX) was derived from normal mouse mammary tissue and exhibits several characteristics distinctive to normal mammary gland epithelial cells (35). COMMA-D cells were cultured in
TABLE
PLAz Activity
I
in Cell-Free Preparations Mammary Carcinoma-Derived
Reaction
condition
Standardb -BSA -CaClx +0.12 mM Nonidet-P40c +0.5 mM sodium deoxycholatec +0.8 mM Chaps’
from Cells
PLA? activity” 31.0 * 1.0 11.8 t 1.6 1.4 + 0.4
0.6 f 0.6 7.6 f 1.0 3.2 + 0.2
Mouse
% activity relative to standard 100
38 5 2 24 10
“Activity is expressed as percentage radiolabeled arachidonic acid released per 2 min per 160 pg protein per milliliter for all assays in the tables. b DMBA-2 cells were harvested in L-l buffer. The reaction mixture contained supernatant fraction (80 ag protein/ml); incubation time was 2 min. All assays contained 2 mM EGTA and 2 mM MgCl,, the “-CaCl;’ sample contained 0.2 mM CaCl, (from the L-l buffer) and thus no free Ca*+ due to excess EGTA. Additions to or omissions from the assay mixture were as indicated above. All other conditions were as described in the Materials and Methods section. c The concentrations of sodium deoxycholate and Chaps are below the CMC, determined in 0.1-0.2 M NaCl, while that of Nonidet-P40 is approximately at the CMC. Higher concentrations of these detergents, i.e., above the CMC, were all inhibitory (data not shown).
FIG. 1. PLA, activity as a function of protein concentration and time. A. Activity (% [3H]arachidonic acid released) as a function of protein concentration per 2-min incubation. B. Activity as a function of time per 80 pg protein/ml. PLAz analyses were performed with a high speed supernatant fraction from MTV-L cells harvested in L-l buffer; incubation time 2 min (A), cell-free protein concentration 80 pg/ml (B). All other procedures were as described in the Materials and Methods section.
Dulbecco’s modified Eagle’s medium-Ham’s F12 (l:l, v/v) supplemented with 2% fetal bovine serum (v/v), 10 pg bovine pituitary extract/ml, and Mite+ serum extender (1:lOOO final dilution) and analyzed at low passage levels (36). Tumor-derived cells were also grown in this medium or in Dulbecco’s modified Eagle’s medium supplemented with 10% fetal bovine serum (v/v) and 3 pg insulin/ml. Cells were washed twice with calPreparation of cell-free fractions. cium, magnesium free phosphate-buffered saline, pH 7.4 (PBS), and once with harvest buffer and then scraped into the harvest buffer. Cells were disrupted by sonication and then centrifuged at 100,000 g for 50 min at 4°C. The particulate fraction was resuspended by sonication in PBS supplemented with 0.22 mM phenylmethylsulfonyl fluoride, 1 mM benzamidine, 2 pM leupeptin. Buffers used for harvesting were: L-l mM CaCl,, 20 mM Hepes, 10 mM ethylene glycol bis(P-amibuffer-l.0 noethylether)N,N,N’,N’-tetraacetic acid (EGTA), 10 mM MgC&, 50 mM NaCl, 1 mM NaNs (pH 7.4) (37); L-11 buffer-11.0 mM CaCl,, 20 mM Hepes, 10 mM EGTA, 10 mM MgCl*, 50 mM NaCl, 1 mM NaNs (pH 7.4); and PBS. All harvest buffers were supplemented with the above protease inhibitors. The protein content of each fraction was determined using the Bradford protein assay reagent of Bio-Rad (Richmond, CA) with bovine serum albumin (BSA) as standard (38). Cell-free samples were stored in 50% glycerol (v/v) at -20°C. PLA, assay. The standard reaction mixture consisted of cell-free sample (in 20 pl buffer/glycerol, l/l, v/v), 1.0 mM CaCl,, 220 mM Tris/ HCl (pH 9.5 at 37”C), 100 mM NaCl, 260 pg fatty acid free BSA/ml, 0.25 mM DTT, and 8.15 pM l-stearoyl-2-[3H]arachidonoyl-sn-glycero3-phosphocholine (0.2 uCi/ml) in a final volume of 50 ~1. Typically the final protein concentration of the cell-free sample in the reaction mixture was either 80 or 160 pg protein/ml, i.e., 4 or 8 pg protein/50-al assay. Lipid substrates were dried under Nz and resuspended by sonication in 1 M Tris/HCl (pH 9.5 at 37°C). Except where indicated, reaction mixtures contained a final free CaZf concentration between 1 and 2 mM, i.e., additional CaCl, was added to the assays when cell-free samples contained excess EGTA relative to CaCI,. Assays were performed, with shaking, at 34°C and terminated by addition of chloroform/methanol (l/2, v/v) containing arachidonic acid, phosphatidylcholine, and dipalmitin. Lysophosphatidylcholine, lysopbosphatidylethanolamine, and
PHOSPHOLIPASE TABLE
A, IN MAMMARY
II
Substrate Specificity of PLAz Activity from Mouse Mammary Carcinoma-Derived Cells Substrate Phosphatidylcholine 2-Arachidonoyl 2-Linoleoyl 2-Oleoyl 2-Palmitoyl Phosphatidylethanolamine 2-Arachidonoyl Phosphatidylinositol 2-Arachidonoyl
PLAr activitya
32.2 6.6 3.5 0.9
+ f f +
2.6b 0.2 0.4 0.1
28.4 k 1.2 0.2 + 0.1
’ Activity is expressed as percentage radiolabeled arachidonic acid released per 2 min per 160 ag protein per milliliter. b PLA, activity in an L-l supernatant fraction from MTV-L cells with 8.15 pM substrate. Assay conditions were 2-min incubations with 80 pg protein/ml for 2-arachidonoyl containing phosphatidylcholine and phosphatidylethanolamine; for all other substrates, the protein concentration in the assay was 160 pg protein/ml and the incubation period was 5 min for phosphatidylcholine containing either a 2-oleoyl or 2linoleoyl group, 10 min for (2-palmitoyl) phosphatidylcholine, and 15 min for (2-arachidonoyl) phosphatidylinositol. No other lipids were tested as substrates. Longer incubation times were employed in assays where there were low levels of hydrolysis in order to more accurately assess activity.
phosphatidic acid were added to the chloroform/methanol, as needed. Lipids were extracted (39) and spotted on the preadsorbent zone of a silica gel TLC plate and chromatographed in benzene/diethyl ether/ ethanol/acetic acid (50/25/2/0.2, by vol). Lipids were visualized with iodine, the phosphatidylcholine and arachidonic acid regions (or other appropriate regions as needed) were scraped from the TLC plates, the lipids were eluted with methanol/Beckman Biosolv-3 (5/2, v/v), and radioactivity was quantified by scintillation spectroscopy. The TLC system used for quantitation of lysophospholipid, phosphatidylethanolamine, and phosphatidylcholine was chloroform/methanol/acetic acid/water (100/60/16/8, by vol) and for phosphatidic acid and phosphatidylcholine quantitation was diethyl ether/acetic acid (100/l, v/v) followed by chromatography in the same direction in chloroform/acetone/methanol/acetic acid/water (50/20/10/10/5, by vol) (40). These chromatography systems were also used to verify the purity of the radiolabeled phospholipid substrates, i.e., cochromatography of radiolabeled and nonradiolabeled phospholipid. Typically the substrates were 98-99% pure by this criterion. Occasionally radiolabeled phospholipids were found to have broken down during storage; such lipids were not used in assays. Assays were performed in quadruplicate, data presented include subtraction of zero time controls. Assays were performed with at least three independent cell harvests. The means and standard deviations are presented for representative experiments.
RESULTS PLAz Activity
in Mammary
Carcinoma-Derived
Cells
PLA:! activity was observed in cell-free preparations from mouse mammary carcinoma-derived cells with lstearoyl-2-[3H]arachidonoyl-sn-glycero-3-phosphocholine as substrate (Table I). This is a substantive activity since release of 31% arachidonic acid (standard assay condition) is equivalent to hydrolysis of 8 nmol substrate per mil-
GLAND-DERIVED
CELLS
FROM
295
MOUSE
ligram protein per minute. There was a requirement for Ca2+ for activity; activity was enhanced by the inclusion of BSA in the reaction mixture and was inhibited by detergents, including Nonidet-P40, sodium deoxycholate, and 3-[(3-cholamidopropyl)-dimethylammonio]-l-propanesulfonate (Chaps) (Table I). When boiled cell-free samples were added to the assay system and incubated for 15 min, no hydrolysis was observed (data not shown). PLAQ activity was linear as a function of time and protein concentration (Fig. 1). PLAP-mediated hydrolysis of lstearoyl-2-[3H]arachidonoyl-sn-glycero-3-phosph~holine results in direct release of [3H]arachidonic acid. [3H]Arachidonic acid could potentially be generated by sequential lytic actions, i.e., phospholipase C and diacylglycerol lipase, with intermediate formation of [3H]diacylglycerol; PLAi and lysoPLAB , with intermediate formation of [3H]lysophosphatidylcholine (2-acyl); and phospholipase D and phosphatidic acid-specific PLA2, with intermediate formation of [3H]phosphatidic acid. Standard assays incubated for 1, 2, 5, and 15 min were evaluated for the formation of these potential intermediates. No [3H]diacylglycerol, [3H]lysophosphatidylcholine (2-acyl), or [3H]phosphatidic acid was observed (data not shown). The substrate specificity of the activity was examined by variation of the fatty acyl group in the sn-2 position of phosphatidylcholine and by variation of the polar head
g20.A 0 4 ii
.a-.’
KlO. 0
0 o-o’
2
x
7
8
9
10
PH
FIG. 2. Hydrolysis of phosphatidylcholine and phosphatidylethanolamine as a function of pH. Activity is indicated as a function of the final pH of the reaction mixture. DTT was added to (0) or omitted from (0) the reaction mixture. Assays were for 2 min with 80 pg protein/ ml of an L-l supernatant fraction from MTV-L cells with 8.15 flM lstearoyl-2-[3H]arachidonoyl-s~-glycero-3-phosphocholine (A) and 8.15 pM l-palmitoyl-2-[“C]arachidonoyl-sn-glycero-3-phosph~thanol~ine (B). Data is presented as activity per 80 pg protein/ml per 2 min. In preliminary experiments, phospholipase C activity was observed at lower pHs (data not shown). Thus PLAz activity at lower pHs was not examined due to the complication of competing reactions.
296
MARION
R. STEINER
TABLE Effect
Harvest
of PLA,
Recentrifugation of L-l supernatant + pellet fractions
buffer*
[3H]Phosphatidylcholine L-11 (Free CaZC) L-l (No free Ca*+)
III
of Ca2+ on Localization
Activity PLA, activity” Supernatant
Particulate
substrate
Recentrifugation, Recentrifugation,
no CaCl, added CaCl, added
1.7 36.2 36.0 3.2
f f f +
0.3c 6.5 5.1 0.0
8.3 1.2 1.0 8.8
+ f + f
0.3 0.1 0.1 1.8
no CaC& added CaCl, added
0.9 30.1 25.6 1.9
iz k f k
0.1 3.2 1.3 0.1
3.5 0.8 0.6 3.9
+ k + k
0.4 0.1 0.0 0.4
[“C]Phosphatidylethanolamine substrated L-11 (Free Cal+) L-l (No free Ca’+) Recentrifugation, Recentrifugation,
a Activity is expressed as percentage radiolabeled arachidonic acid released per 2 min per 160 pg protein per milliliter. * MTV-L cells were harvested in L-l buffer, i.e., a buffer which does not contain free Ca’+, or L-11 buffer, a buffer which contains free Ca’+. For recentrifugation of L-l samples, the particulate fraction was resuspended in the supernatant fraction and, where indicated, CaCl, was added (11 mM final concentration). ’ Assays were performed with 80 fig protein/ml, L-l supernatant fraction and L-l supernatant fraction after recentrifugation (no CaCl, added); with 160 pg protein/ml, all other samples. Reactions times were 2 min, supernatant fractions; 5 min, particulate fractions. All other reaction conditions were as described in the Materials and Methods section including the addition of sufficient CaCl, so that all assays contained free Ca’+. dThe reaction mixture contained 10 ~1 1.0 M Tris/HCl (pH 8.5 at 37’C) plus 1 ~1 1.0 M Tris/HCl (pH 9.5 at 37°C) for assays with [“C]phosphatidylethanolamine (8.15 PM).
group ofphospholipids with an arachidonoyl group in the sn-2 position. PLAP activity was highest when the fatty acyl moiety in the sn-2 position was an arachidonoyl group as compared to other fatty acyl substituents (Table II). Preferential hydrolysis of arachidonoyl containing phosphatidylcholine was also observed in studies with lipid mixtures. For example, hydrolysis of (2-arachidonoyl)phosphatidylcholine was eightfold higher than hydrolysis of (2-palmitoyl)phosphatidylcholine in assays with equimolar mixtures of the two phosphatidylcholines (final lipid concentration 49 PM). Significant PLAz activity was observed with arachidonoyl containing phosphatidylcholine or phosphatidylethanolamine as substrates whereas (2-arachidonoyl)phosphatidylinositol was not an effective substrate. The pH profiles for hydrolysis of arachidonoyl containing phosphatidylcholine and phosphatidylethanolamine differed (Fig. 2). When DTT was omitted from the assay mixture, there was a greater difference in activity as a function of pH between the two substrates. Other reaction parameters were similar for hydrolysis of these two substrates, e.g., when CaC12 was omitted from the reaction mixture, as described in Table I, only 1.7% of the phosphatidylethanolamine was hydrolyzed (per 2 min per 160 Kg protein/ml). Localization
of Enzyme Activity
Some PLAzs are membrane associated whereas others are found in the soluble fraction following centrifugation
(e.g., (41-43)). When mammary carcinoma-derived cells were harvested in a buffer containing free Ca2+ (L-11 buffer), significant activity was observed in the particulate fraction (Table III). In contrast, when these cells were harvested in a buffer containing EGTA (no free Ca2+, L-l buffer), activity was observed primarily in the supernatant fraction following high speed centrifugation. These results suggest calcium-mediated binding of PLA2 to membranes. This was further supported by the observation that activity initially observed in the L-l supernatant fraction could be recovered in the particulate fraction upon recentrifugation of the supernatant fraction plus free Ca2+ and membranes (Table III). The maximum activity observed in the supernatant fraction was significantly higher than that in the particulate fraction. The reduction in the specific activity of the substrate in the particulate fraction assays, due to endogenous substrate, likely contributes to the lower apparent PLA2 activity in this fraction; however, additional factors may also affect PLA2 activity in the particulate fraction, such as other lipids. Cells were harvested in PBS in order to determine the localization of PLA2 activity under limiting Ca2+ concentrations, i.e., less than 1 j.&M (25,44). Both the particulate and the supernatant fractions from cells harvested in PBS contained significant PLA2 activity (Table IV). In addition, both fractions appeared to contain sufficient free Ca2+ for PLA, activity so that a Ca2+ requirement for hydrolysis could only be demonstrated by the addition of EGTA to the reaction mixture.
PHOSPHOLIPASE
A, IN
MAMMARY
TABLE IV
PLAP: Localization and Activity in the Absence of Exogenously Added CaCl* PLA, activity’ Assay condition 1.0 mM CaClx (standard)’ no CaClx added 1.0 mM EGTA, no CaClx added 1.0 mM EGTA + 2.0 mM CaCl,
Particulate
Supernatant 17.3 +- 2.5 15.1 + 0.8
5.1 + 0.5 5.3 2 0.3
0.0 * 0.2 17.7 2 2.4
-0.1 + 0.0 5.1 zk 0.2
’ Activity is expressed as percentage radiolabeled arachidonic acid released per 2 min per 160 pg protein per milliliter. b MTV-L cells were harvested in PBS supplemented with protease inhibitors. The free Cax’ concentrations of PBS homogenates were less than 0.4 pM (as measured with either a final concentration of 2.5 or 5 PM fura- in different analyses; (64)). The assay incubation period was 2 min for the supematant fraction and 5 min for the particulate fraction; both fractions were assayed at a protein concentration of 160 pg/ml.
PLAz Activity
in Normal Gland-Derived
Cells
The question of PLAz activity in normal gland- versus tumor-derived cells was next addressed. Tumor cell activity was more than double that of normal cells (Table V). Comparisons of PLAz activities from normal versus DMBA-2 or MTV-L cells, by Student’s t test, gave a p value of less than 0.001. The normal gland-derived cell activity was similar to the tumor-derived cell activity with respect to substrate specificity and localization as a function of Ca2+ concentration (Table V), as well as other properties, e.g., requirement for Ca2+ for activity (data not shown). Thus the major difference between normal and tumor-derived cells with respect to PLAB activity is the significantly higher level of activity in tumor-derived cells. DISCUSSION
A calcium-dependent PLAZ activity(ies) which exhibits specificity for hydrolysis of phosphatidylcholine and phosphatidylethanolamine, with an arachidonoyl group in the sn-2 position, has been observed in cell-free extracts from mouse mammary gland-derived cells. PLAz activity differs as a function of pH for the two substrates. This pH difference may relate to differences between the two substrates as a function of pH or the presence of specific enzymatic activities for each substrate. In addition to the differential alteration in charge as a function of pH with the two substrates, their physical forms may also differ, The mammary PLAz activity(ies) is similar to other nonpancreatic PLAzs with respect to stimulation by inclusion of BSA in the reaction mixture, a Ca2+ requirement for activity, and activity at alkaline pHs (41-47). The concentration of Ca2+ required for activity of the mammary
GLAND-DERIVED
CELLS
FROM
297
MOUSE
gland PLAz may be physiologically relevant since activity could be observed in the absence of exogenously added Ca+2 while addition of EGTA was necessary to demonstrate the Ca2+ requirement for activity. The localization of the mammary PLAz activity is dependent upon the presence or absence of free Ca2+. Localization of other PLA2s may also be dependent upon the level of free Ca2+ since mesangial cell and platelet PLAP activities were higher in supernatant fractions from homogenates prepared in buffers containing divalent cation chelators versus those prepared in buffers containing free Ca2+ (45,48). Quite recently, a Ca2+-dependent membrane association of a macrophage PLA2 and a brain cytosolic PLA2 have been demonstrated (49,50). However, association of PLA,s with membranes, only in the presence of free Ca2+, is not a characteristic of all PLA,s since membrane fraction-associated PLAz activity has been observed following preparation of cell-free fractions in an EGTA-EDTA containing buffer (51,52). The observation that localization of mammary PLA2 activity can be modulated by free Ca2+ raises the possibility that this PLA2 may be translocated in response to changes in intracellular
TABLE
V
Substrate Specificity (A) and Localization (B) of PLA2 Activity
in Cell-Free Preparations from Gland-Derived Cells A. Substrate
Normal
specificity”
Substrate
PLAB activityb
Phosphatidylcholine 2-Arachidonoyl 2-Linoleoyl 2-Oleoyl 2-Palmitoyl Phosphatidylethanolamine 2-Arachidonoyl Phosphatidylinositol 2-Arachidonoyl
12.7 1.2 1.1 0.3
f 2.5 f 0.2 + 0.1 XL0.1
7.0 f 0.7 0.04 +- 0.02
B. Localization’ PLAx activityb Harvest buffer [3H]Phosphatidylcholine substrate L-l L-11 [i4C]Phosphatidylethanolamine substrate L-l L-11
Supernatant
Particulate
10.0 k 0.7 0.2 +- 0.2
0.6 IL 0.1 2.8 ? 0.3
8.3 XL0.6 0.2 f 0.1
0.4 2 0.0 0.9 f 0.2
a COMMA-D cells were harvested in L-l buffer and the supernatant fraction (160 fig protein/ml) was analyzed as described in Table II. b Activity is expressed as percentage radiolabeled arachidonic acid released per 2 min per 160 pg protein per milliliter. ‘COMMA-D cells were harvested in L-l buffer or L-11 buffer and assayed at 160 ag protein/ml as described in Table III.
298
MARION
Ca2+ concentrations. Translocation of other enzymes, such as protein kinase C, 5-lipoxygenase, and CTP:phosphocholine cytidylyltransferase, is important in the regulation of the activity of these enzymes (53-55). Moreover, translocation of a macrophage PLAB has been observed in response to treatment of cells with diacylglycerol (56). Mouse mammary carcinoma-derived cells had a higher PLA2 activity than normal gland-derived cells with either arachidonoyl containing phosphatidylcholine or phosphatidylethanolamine as a substrate. This increased activity in tumor cell preparations may result in increased eicosanoid synthesis. The increased activity, with either substrate, in tumor versus normal cell preparations would suggest that there is one enzyme which can hydrolyze both substrates or that there is coordinate regulation of activity of two enzymes. The increased tumor cell PLA2 activity may be due to an increased amount of enzyme, increased specific activity of the enzyme, e.g., due to changes in post-translational modifications, or alterations in inhibitory or stimulatory factors. Stimulation of PLA2 activity, in other cell systems, was observed in response to a range of factors including components of signal transduction pathways, such as diacylglycerol and G proteins (57-62). Moreover, another GTP binding protein, the ras oncogene product, was associated with elevated PLA2 activity (16, 17, 63). The combined studies suggest the intriguing possibility that alterations in PLA2 activity may be a feature of a range of neoplastic systems and may relate to alterations in signal transduction systems in neoplasia. ACKNOWLEDGMENTS This work was supported by Grant CA25730 and Biomedical Research Support Grant RR05374 from the National Institutes of Health. The excellent technical assistance of Teresa Perrone is gratefully acknowledged. Dr. Jesse Sisken and Terri Newcombe are thanked for the fura2 analyses.
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PHOSPHOLIPASE
AZ IN MAMMARY
GLAND-DERIVED
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Z.&t. 136.298-300. 48. Gronich, J. H., Bonventre, J. V., and Nemenoff, R. A. (1988) J. Btil. C&m. 263,16,645-16,651. 49. Channon, J. Y., and Leslie, C. C. (1990) J. Biol. Chem. 265,54095413. 50. Yoshihara, Y., and Watanbe, Y. (1990) Biochem. Biophys. Res. Commun. 170.484-490. 51. De Winter, J. M., Vianen, G. M., and Van den Bosch, H. (1982) Biochim. Biophys. Acta 712, 332-341. 52. Ross, M. I., Deems, R. A., Jesaitis, A. J., Dennis, E. A., and Ulevitch, R. J. (1985) Arch. B&hem. Biophys. 238, 247-258. 53. Kraft, A. S., and Anderson, W. B. (1983) Nature (London) 301,
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54. Pelech, S. L., Cook, H. W., Paddon, H. B., and Vance, D. E. (1984) B&him. Biophys. Acta 795,433-440. 55. Rouzer, C. A., and Samuelsson, USA 84, 7393-7397.
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R. M., Checani, G. C., and Deykin,
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OF BIOCHEMISTRY
AND
BIOPHYSICS
Vol. 286, No. 1, April, pp. 293-299, 1991
Localization and Characterization of Phospholipase in Mouse Mammary Gland-Derived Cells Marion
A,
R. Steiner
Department of Microbiology and Immunology, Medical Center, Room MS 413, University of Kentucky, Lexington, Kentucky 40536
Received August 27, 1990, and in revised form December 6, 1990
Phospholipase A2 (PLA2) can participate in the regulation of eicosanoid biosynthesis via PLAz-mediated control of the release of arachidonic acid from phospholipids. Arachidonoyl-hydrolyzing PLA,s were examined in cells from normal mouse mammary glands and mammary carcinomas. Tumor-derived cells exhibited significant PLAz activity(ies) with arachidonoyl containing phosphatidylcholine and phosphatidylethanolamine as substrates in cell-free assays. In contrast, arachidonoyl containing phosphatidylinositol was a poor substrate. When phosphatidylcholines with varying sn-2 fatty acyl groups were tested as substrates, activity was highest with the arachidonoyl containing lipid. The pH profiles for hydrolysis of phosphatidylcholine and phosphatidylethanolamine differed; all other aspects of PLA,-mediated hydrolysis of these two substrates were similar including a Ca2+ requirement for activity. Moreover, Ca2+ affected the subcellular localization of the enzyme activity. Activity was predominately in the supernatant fraction when cells were harvested in an EGTA (ethylene glycol bis(@-aminoethylether)-N,N,N’,N’-tetraacetic acid) containing buffer and largely in the particulate fraction when cells were harvested in a buffer containing free Ca2+. The localization of activity could be modulated from the supernatant fraction to the particulate fraction by recentrifugation in the presence of Ca2+. Normal gland-derived cells contained a PLAz activity with properties similar to those of the tumor-derived cells. There was a significant difference in the level of activity in the normal versus tumor cells, the normal gland-derived cells had less than half the PLAz activity of the carcinomaderived cells. o lee1 Academic PPXSS, IN.
The arachidonic acid metabolites, prostaglandins, have been implicated in the development of primary mammary carcinomas and in metastasis of mammary neoplasias (l8). In addition, arachidonic acid metabolites appear to be important in normal gland function since eicosanoids 0003-9861/91$3.00 Copyright 0 1991 by Academic Press, All rights of reproduction in any form
stimulate normal cell proliferation (9). Regulation of eicosanoid synthesis is mediated in part via control of the release of arachidonic acid from glycerolipids (10). Phospholipase A2 (PLAs)’ has been implicated in the regulation of eicosanoid synthesis via its involvement in the release of arachidonic acid from phospholipids (10). For example, increased PLAs activity has been associated with increased eicosanoid synthesis in f-Met-Leu-Phe-stimulated macrophages and interleukin l-treated fibroblasts and chondrocytes (11-13). The status of PLAz activity in neoplasia has been examined in only a limited number of studies. Cell-free preparations from rat mammary carcinomas and gastric carcinomatous mucosa exhibited an increased PLAs activity as compared to preparations from normal glands and normal mucosa, respectively (14, 15). In addition, PLAs activity was higher in cultured rat fibroblasts transfected with the ras oncogene than in nontransfected control cells (16, 17). Specificity of activity with respect to the fatty acyl group in the sn-2 position and PLA2 properties were not addressed in these studies. PLA,s participate in a range of functions including phospholipid degradation, phospholipid remodeling, and regulation of free arachidonic acid levels (reviewed in (1820)), hence PLAss involved in different functions are likely to have different characteristics. With respect to specificity for an arachidonoyl group in the sn-2 position, three classes of PLAss have been observed: (i) those exhibiting a specificity for arachidonoyl groups (21-26), (ii) those exhibiting no fatty acyl specificity (27-30), and (iii) those which preferentially hydrolyze phospholipids containing fatty acyl groups other than an arachidonoyl group (31, 32). The present study was undertaken to ascertain if mouse mammary gland-derived cells have a PLA, activ1 Abbreviations used: BSA, bovine serum albumin; Chaps, 3-[(3-cholamidopropyl)-dimethylammonio]-l-propanesulfonate; DTT, dithiothreitol; EGTA, ethylene glycol his@-aminoethylether)-N,N,N:N’-tetraacetic acid; PBS, magnesium and calcium free phosphate-buffered saline; PLA, phospholipase A; TLC, thin layer chromatography. 293
Inc. reserved.
294
MARION
R. STEINER
ity(ies) which preferentially hydrolyzes phospholipids containing arachidonoyl residues, to characterize this activity, and to determine the status of this PLAz activity in mammary carcinoma-derived cells as compared to normal gland-derived cells. MATERIALS
AND
METHODS
I-Stearoyl-2[3H] arachidonoyl-sn-glycero-3-phosphoMaterids. choline (91 Ci/mmol), l-palmitoyl-2-[‘“Clarachidonoyl-sn-glycero-3phosphoethanolamine (52 mCi/mmol), l-stearoyl-2-[“Clarachidonoylsn-glycero-3-phosphoinositol (30 mCi/mmol), 1-palmitoyl-2-[sH] palmitoyl-sn-glycero-3-phosphocholine (57 Ci/mmol), 1-palmitoyl-2[“C]linoleoyl-sn-glycero-3-phosphocholine (58 mCi/mmol), and l-palmitoyl-2-[1”C]oleoyl-sn-glycero-3-phosphocholine (53 mCi/mmol) were obtained from DuPont-NEN Research Products (Boston, MA). The source of l-stearoyl-2-arachidonoyl-sn-glycero-3-phosphocholine was Avanti Polar Lipids (Birmingham, AL); Hepes, dithiothreitol (DTT), and leupeptin were from Calbiochem (San Diego, CA); all other biochemicals used in the PLAx assay were from Sigma Chemical Co. (St. Louis, MO). Bovine pituitary extract and Mite+ serum extender were obtained from Collaborative Research, Inc (Bedford, MA); other tissue culture reagents were obtained from GIBCO Laboratories (Grand Island, NY) and Sigma Chemical Co. Thin layer chromatography (TLC) plates were from Analtech (Newark, DE); Beckman Biosolv-3 was from Beckman Instruments (Irvine, CA). Cells and cell culture. The MTV-L/BALB Cl 2 and DMBA-P/BALB Cl 4 cell lines were derived from mouse mammary carcinomas which had viral and chemical carcinogen etiologies respectively (33, 34). The COMMA-D cell line (generously provided by Dr. D. Medina, Baylor College of Medicine, Houston, TX) was derived from normal mouse mammary tissue and exhibits several characteristics distinctive to normal mammary gland epithelial cells (35). COMMA-D cells were cultured in
TABLE
PLAz Activity
I
in Cell-Free Preparations Mammary Carcinoma-Derived
Reaction
condition
Standardb -BSA -CaClx +0.12 mM Nonidet-P40c +0.5 mM sodium deoxycholatec +0.8 mM Chaps’
from Cells
PLA? activity” 31.0 * 1.0 11.8 t 1.6 1.4 + 0.4
0.6 f 0.6 7.6 f 1.0 3.2 + 0.2
Mouse
% activity relative to standard 100
38 5 2 24 10
“Activity is expressed as percentage radiolabeled arachidonic acid released per 2 min per 160 pg protein per milliliter for all assays in the tables. b DMBA-2 cells were harvested in L-l buffer. The reaction mixture contained supernatant fraction (80 ag protein/ml); incubation time was 2 min. All assays contained 2 mM EGTA and 2 mM MgCl,, the “-CaCl;’ sample contained 0.2 mM CaCl, (from the L-l buffer) and thus no free Ca*+ due to excess EGTA. Additions to or omissions from the assay mixture were as indicated above. All other conditions were as described in the Materials and Methods section. c The concentrations of sodium deoxycholate and Chaps are below the CMC, determined in 0.1-0.2 M NaCl, while that of Nonidet-P40 is approximately at the CMC. Higher concentrations of these detergents, i.e., above the CMC, were all inhibitory (data not shown).
FIG. 1. PLA, activity as a function of protein concentration and time. A. Activity (% [3H]arachidonic acid released) as a function of protein concentration per 2-min incubation. B. Activity as a function of time per 80 pg protein/ml. PLAz analyses were performed with a high speed supernatant fraction from MTV-L cells harvested in L-l buffer; incubation time 2 min (A), cell-free protein concentration 80 pg/ml (B). All other procedures were as described in the Materials and Methods section.
Dulbecco’s modified Eagle’s medium-Ham’s F12 (l:l, v/v) supplemented with 2% fetal bovine serum (v/v), 10 pg bovine pituitary extract/ml, and Mite+ serum extender (1:lOOO final dilution) and analyzed at low passage levels (36). Tumor-derived cells were also grown in this medium or in Dulbecco’s modified Eagle’s medium supplemented with 10% fetal bovine serum (v/v) and 3 pg insulin/ml. Cells were washed twice with calPreparation of cell-free fractions. cium, magnesium free phosphate-buffered saline, pH 7.4 (PBS), and once with harvest buffer and then scraped into the harvest buffer. Cells were disrupted by sonication and then centrifuged at 100,000 g for 50 min at 4°C. The particulate fraction was resuspended by sonication in PBS supplemented with 0.22 mM phenylmethylsulfonyl fluoride, 1 mM benzamidine, 2 pM leupeptin. Buffers used for harvesting were: L-l mM CaCl,, 20 mM Hepes, 10 mM ethylene glycol bis(P-amibuffer-l.0 noethylether)N,N,N’,N’-tetraacetic acid (EGTA), 10 mM MgC&, 50 mM NaCl, 1 mM NaNs (pH 7.4) (37); L-11 buffer-11.0 mM CaCl,, 20 mM Hepes, 10 mM EGTA, 10 mM MgCl*, 50 mM NaCl, 1 mM NaNs (pH 7.4); and PBS. All harvest buffers were supplemented with the above protease inhibitors. The protein content of each fraction was determined using the Bradford protein assay reagent of Bio-Rad (Richmond, CA) with bovine serum albumin (BSA) as standard (38). Cell-free samples were stored in 50% glycerol (v/v) at -20°C. PLA, assay. The standard reaction mixture consisted of cell-free sample (in 20 pl buffer/glycerol, l/l, v/v), 1.0 mM CaCl,, 220 mM Tris/ HCl (pH 9.5 at 37”C), 100 mM NaCl, 260 pg fatty acid free BSA/ml, 0.25 mM DTT, and 8.15 pM l-stearoyl-2-[3H]arachidonoyl-sn-glycero3-phosphocholine (0.2 uCi/ml) in a final volume of 50 ~1. Typically the final protein concentration of the cell-free sample in the reaction mixture was either 80 or 160 pg protein/ml, i.e., 4 or 8 pg protein/50-al assay. Lipid substrates were dried under Nz and resuspended by sonication in 1 M Tris/HCl (pH 9.5 at 37°C). Except where indicated, reaction mixtures contained a final free CaZf concentration between 1 and 2 mM, i.e., additional CaCl, was added to the assays when cell-free samples contained excess EGTA relative to CaCI,. Assays were performed, with shaking, at 34°C and terminated by addition of chloroform/methanol (l/2, v/v) containing arachidonic acid, phosphatidylcholine, and dipalmitin. Lysophosphatidylcholine, lysopbosphatidylethanolamine, and
PHOSPHOLIPASE TABLE
A, IN MAMMARY
II
Substrate Specificity of PLAz Activity from Mouse Mammary Carcinoma-Derived Cells Substrate Phosphatidylcholine 2-Arachidonoyl 2-Linoleoyl 2-Oleoyl 2-Palmitoyl Phosphatidylethanolamine 2-Arachidonoyl Phosphatidylinositol 2-Arachidonoyl
PLAr activitya
32.2 6.6 3.5 0.9
+ f f +
2.6b 0.2 0.4 0.1
28.4 k 1.2 0.2 + 0.1
’ Activity is expressed as percentage radiolabeled arachidonic acid released per 2 min per 160 ag protein per milliliter. b PLA, activity in an L-l supernatant fraction from MTV-L cells with 8.15 pM substrate. Assay conditions were 2-min incubations with 80 pg protein/ml for 2-arachidonoyl containing phosphatidylcholine and phosphatidylethanolamine; for all other substrates, the protein concentration in the assay was 160 pg protein/ml and the incubation period was 5 min for phosphatidylcholine containing either a 2-oleoyl or 2linoleoyl group, 10 min for (2-palmitoyl) phosphatidylcholine, and 15 min for (2-arachidonoyl) phosphatidylinositol. No other lipids were tested as substrates. Longer incubation times were employed in assays where there were low levels of hydrolysis in order to more accurately assess activity.
phosphatidic acid were added to the chloroform/methanol, as needed. Lipids were extracted (39) and spotted on the preadsorbent zone of a silica gel TLC plate and chromatographed in benzene/diethyl ether/ ethanol/acetic acid (50/25/2/0.2, by vol). Lipids were visualized with iodine, the phosphatidylcholine and arachidonic acid regions (or other appropriate regions as needed) were scraped from the TLC plates, the lipids were eluted with methanol/Beckman Biosolv-3 (5/2, v/v), and radioactivity was quantified by scintillation spectroscopy. The TLC system used for quantitation of lysophospholipid, phosphatidylethanolamine, and phosphatidylcholine was chloroform/methanol/acetic acid/water (100/60/16/8, by vol) and for phosphatidic acid and phosphatidylcholine quantitation was diethyl ether/acetic acid (100/l, v/v) followed by chromatography in the same direction in chloroform/acetone/methanol/acetic acid/water (50/20/10/10/5, by vol) (40). These chromatography systems were also used to verify the purity of the radiolabeled phospholipid substrates, i.e., cochromatography of radiolabeled and nonradiolabeled phospholipid. Typically the substrates were 98-99% pure by this criterion. Occasionally radiolabeled phospholipids were found to have broken down during storage; such lipids were not used in assays. Assays were performed in quadruplicate, data presented include subtraction of zero time controls. Assays were performed with at least three independent cell harvests. The means and standard deviations are presented for representative experiments.
RESULTS PLAz Activity
in Mammary
Carcinoma-Derived
Cells
PLA:! activity was observed in cell-free preparations from mouse mammary carcinoma-derived cells with lstearoyl-2-[3H]arachidonoyl-sn-glycero-3-phosphocholine as substrate (Table I). This is a substantive activity since release of 31% arachidonic acid (standard assay condition) is equivalent to hydrolysis of 8 nmol substrate per mil-
GLAND-DERIVED
CELLS
FROM
295
MOUSE
ligram protein per minute. There was a requirement for Ca2+ for activity; activity was enhanced by the inclusion of BSA in the reaction mixture and was inhibited by detergents, including Nonidet-P40, sodium deoxycholate, and 3-[(3-cholamidopropyl)-dimethylammonio]-l-propanesulfonate (Chaps) (Table I). When boiled cell-free samples were added to the assay system and incubated for 15 min, no hydrolysis was observed (data not shown). PLAQ activity was linear as a function of time and protein concentration (Fig. 1). PLAP-mediated hydrolysis of lstearoyl-2-[3H]arachidonoyl-sn-glycero-3-phosph~holine results in direct release of [3H]arachidonic acid. [3H]Arachidonic acid could potentially be generated by sequential lytic actions, i.e., phospholipase C and diacylglycerol lipase, with intermediate formation of [3H]diacylglycerol; PLAi and lysoPLAB , with intermediate formation of [3H]lysophosphatidylcholine (2-acyl); and phospholipase D and phosphatidic acid-specific PLA2, with intermediate formation of [3H]phosphatidic acid. Standard assays incubated for 1, 2, 5, and 15 min were evaluated for the formation of these potential intermediates. No [3H]diacylglycerol, [3H]lysophosphatidylcholine (2-acyl), or [3H]phosphatidic acid was observed (data not shown). The substrate specificity of the activity was examined by variation of the fatty acyl group in the sn-2 position of phosphatidylcholine and by variation of the polar head
g20.A 0 4 ii
.a-.’
KlO. 0
0 o-o’
2
x
7
8
9
10
PH
FIG. 2. Hydrolysis of phosphatidylcholine and phosphatidylethanolamine as a function of pH. Activity is indicated as a function of the final pH of the reaction mixture. DTT was added to (0) or omitted from (0) the reaction mixture. Assays were for 2 min with 80 pg protein/ ml of an L-l supernatant fraction from MTV-L cells with 8.15 flM lstearoyl-2-[3H]arachidonoyl-s~-glycero-3-phosphocholine (A) and 8.15 pM l-palmitoyl-2-[“C]arachidonoyl-sn-glycero-3-phosph~thanol~ine (B). Data is presented as activity per 80 pg protein/ml per 2 min. In preliminary experiments, phospholipase C activity was observed at lower pHs (data not shown). Thus PLAz activity at lower pHs was not examined due to the complication of competing reactions.
296
MARION
R. STEINER
TABLE Effect
Harvest
of PLA,
Recentrifugation of L-l supernatant + pellet fractions
buffer*
[3H]Phosphatidylcholine L-11 (Free CaZC) L-l (No free Ca*+)
III
of Ca2+ on Localization
Activity PLA, activity” Supernatant
Particulate
substrate
Recentrifugation, Recentrifugation,
no CaCl, added CaCl, added
1.7 36.2 36.0 3.2
f f f +
0.3c 6.5 5.1 0.0
8.3 1.2 1.0 8.8
+ f + f
0.3 0.1 0.1 1.8
no CaC& added CaCl, added
0.9 30.1 25.6 1.9
iz k f k
0.1 3.2 1.3 0.1
3.5 0.8 0.6 3.9
+ k + k
0.4 0.1 0.0 0.4
[“C]Phosphatidylethanolamine substrated L-11 (Free Cal+) L-l (No free Ca’+) Recentrifugation, Recentrifugation,
a Activity is expressed as percentage radiolabeled arachidonic acid released per 2 min per 160 pg protein per milliliter. * MTV-L cells were harvested in L-l buffer, i.e., a buffer which does not contain free Ca’+, or L-11 buffer, a buffer which contains free Ca’+. For recentrifugation of L-l samples, the particulate fraction was resuspended in the supernatant fraction and, where indicated, CaCl, was added (11 mM final concentration). ’ Assays were performed with 80 fig protein/ml, L-l supernatant fraction and L-l supernatant fraction after recentrifugation (no CaCl, added); with 160 pg protein/ml, all other samples. Reactions times were 2 min, supernatant fractions; 5 min, particulate fractions. All other reaction conditions were as described in the Materials and Methods section including the addition of sufficient CaCl, so that all assays contained free Ca’+. dThe reaction mixture contained 10 ~1 1.0 M Tris/HCl (pH 8.5 at 37’C) plus 1 ~1 1.0 M Tris/HCl (pH 9.5 at 37°C) for assays with [“C]phosphatidylethanolamine (8.15 PM).
group ofphospholipids with an arachidonoyl group in the sn-2 position. PLAP activity was highest when the fatty acyl moiety in the sn-2 position was an arachidonoyl group as compared to other fatty acyl substituents (Table II). Preferential hydrolysis of arachidonoyl containing phosphatidylcholine was also observed in studies with lipid mixtures. For example, hydrolysis of (2-arachidonoyl)phosphatidylcholine was eightfold higher than hydrolysis of (2-palmitoyl)phosphatidylcholine in assays with equimolar mixtures of the two phosphatidylcholines (final lipid concentration 49 PM). Significant PLAz activity was observed with arachidonoyl containing phosphatidylcholine or phosphatidylethanolamine as substrates whereas (2-arachidonoyl)phosphatidylinositol was not an effective substrate. The pH profiles for hydrolysis of arachidonoyl containing phosphatidylcholine and phosphatidylethanolamine differed (Fig. 2). When DTT was omitted from the assay mixture, there was a greater difference in activity as a function of pH between the two substrates. Other reaction parameters were similar for hydrolysis of these two substrates, e.g., when CaC12 was omitted from the reaction mixture, as described in Table I, only 1.7% of the phosphatidylethanolamine was hydrolyzed (per 2 min per 160 Kg protein/ml). Localization
of Enzyme Activity
Some PLAzs are membrane associated whereas others are found in the soluble fraction following centrifugation
(e.g., (41-43)). When mammary carcinoma-derived cells were harvested in a buffer containing free Ca2+ (L-11 buffer), significant activity was observed in the particulate fraction (Table III). In contrast, when these cells were harvested in a buffer containing EGTA (no free Ca2+, L-l buffer), activity was observed primarily in the supernatant fraction following high speed centrifugation. These results suggest calcium-mediated binding of PLA2 to membranes. This was further supported by the observation that activity initially observed in the L-l supernatant fraction could be recovered in the particulate fraction upon recentrifugation of the supernatant fraction plus free Ca2+ and membranes (Table III). The maximum activity observed in the supernatant fraction was significantly higher than that in the particulate fraction. The reduction in the specific activity of the substrate in the particulate fraction assays, due to endogenous substrate, likely contributes to the lower apparent PLA2 activity in this fraction; however, additional factors may also affect PLA2 activity in the particulate fraction, such as other lipids. Cells were harvested in PBS in order to determine the localization of PLA2 activity under limiting Ca2+ concentrations, i.e., less than 1 j.&M (25,44). Both the particulate and the supernatant fractions from cells harvested in PBS contained significant PLA2 activity (Table IV). In addition, both fractions appeared to contain sufficient free Ca2+ for PLA, activity so that a Ca2+ requirement for hydrolysis could only be demonstrated by the addition of EGTA to the reaction mixture.
PHOSPHOLIPASE
A, IN
MAMMARY
TABLE IV
PLAP: Localization and Activity in the Absence of Exogenously Added CaCl* PLA, activity’ Assay condition 1.0 mM CaClx (standard)’ no CaClx added 1.0 mM EGTA, no CaClx added 1.0 mM EGTA + 2.0 mM CaCl,
Particulate
Supernatant 17.3 +- 2.5 15.1 + 0.8
5.1 + 0.5 5.3 2 0.3
0.0 * 0.2 17.7 2 2.4
-0.1 + 0.0 5.1 zk 0.2
’ Activity is expressed as percentage radiolabeled arachidonic acid released per 2 min per 160 pg protein per milliliter. b MTV-L cells were harvested in PBS supplemented with protease inhibitors. The free Cax’ concentrations of PBS homogenates were less than 0.4 pM (as measured with either a final concentration of 2.5 or 5 PM fura- in different analyses; (64)). The assay incubation period was 2 min for the supematant fraction and 5 min for the particulate fraction; both fractions were assayed at a protein concentration of 160 pg/ml.
PLAz Activity
in Normal Gland-Derived
Cells
The question of PLAz activity in normal gland- versus tumor-derived cells was next addressed. Tumor cell activity was more than double that of normal cells (Table V). Comparisons of PLAz activities from normal versus DMBA-2 or MTV-L cells, by Student’s t test, gave a p value of less than 0.001. The normal gland-derived cell activity was similar to the tumor-derived cell activity with respect to substrate specificity and localization as a function of Ca2+ concentration (Table V), as well as other properties, e.g., requirement for Ca2+ for activity (data not shown). Thus the major difference between normal and tumor-derived cells with respect to PLAB activity is the significantly higher level of activity in tumor-derived cells. DISCUSSION
A calcium-dependent PLAZ activity(ies) which exhibits specificity for hydrolysis of phosphatidylcholine and phosphatidylethanolamine, with an arachidonoyl group in the sn-2 position, has been observed in cell-free extracts from mouse mammary gland-derived cells. PLAz activity differs as a function of pH for the two substrates. This pH difference may relate to differences between the two substrates as a function of pH or the presence of specific enzymatic activities for each substrate. In addition to the differential alteration in charge as a function of pH with the two substrates, their physical forms may also differ, The mammary PLAz activity(ies) is similar to other nonpancreatic PLAzs with respect to stimulation by inclusion of BSA in the reaction mixture, a Ca2+ requirement for activity, and activity at alkaline pHs (41-47). The concentration of Ca2+ required for activity of the mammary
GLAND-DERIVED
CELLS
FROM
297
MOUSE
gland PLAz may be physiologically relevant since activity could be observed in the absence of exogenously added Ca+2 while addition of EGTA was necessary to demonstrate the Ca2+ requirement for activity. The localization of the mammary PLAz activity is dependent upon the presence or absence of free Ca2+. Localization of other PLA2s may also be dependent upon the level of free Ca2+ since mesangial cell and platelet PLAP activities were higher in supernatant fractions from homogenates prepared in buffers containing divalent cation chelators versus those prepared in buffers containing free Ca2+ (45,48). Quite recently, a Ca2+-dependent membrane association of a macrophage PLA2 and a brain cytosolic PLA2 have been demonstrated (49,50). However, association of PLA,s with membranes, only in the presence of free Ca2+, is not a characteristic of all PLA,s since membrane fraction-associated PLAz activity has been observed following preparation of cell-free fractions in an EGTA-EDTA containing buffer (51,52). The observation that localization of mammary PLA2 activity can be modulated by free Ca2+ raises the possibility that this PLA2 may be translocated in response to changes in intracellular
TABLE
V
Substrate Specificity (A) and Localization (B) of PLA2 Activity
in Cell-Free Preparations from Gland-Derived Cells A. Substrate
Normal
specificity”
Substrate
PLAB activityb
Phosphatidylcholine 2-Arachidonoyl 2-Linoleoyl 2-Oleoyl 2-Palmitoyl Phosphatidylethanolamine 2-Arachidonoyl Phosphatidylinositol 2-Arachidonoyl
12.7 1.2 1.1 0.3
f 2.5 f 0.2 + 0.1 XL0.1
7.0 f 0.7 0.04 +- 0.02
B. Localization’ PLAx activityb Harvest buffer [3H]Phosphatidylcholine substrate L-l L-11 [i4C]Phosphatidylethanolamine substrate L-l L-11
Supernatant
Particulate
10.0 k 0.7 0.2 +- 0.2
0.6 IL 0.1 2.8 ? 0.3
8.3 XL0.6 0.2 f 0.1
0.4 2 0.0 0.9 f 0.2
a COMMA-D cells were harvested in L-l buffer and the supernatant fraction (160 fig protein/ml) was analyzed as described in Table II. b Activity is expressed as percentage radiolabeled arachidonic acid released per 2 min per 160 pg protein per milliliter. ‘COMMA-D cells were harvested in L-l buffer or L-11 buffer and assayed at 160 ag protein/ml as described in Table III.
298
MARION
Ca2+ concentrations. Translocation of other enzymes, such as protein kinase C, 5-lipoxygenase, and CTP:phosphocholine cytidylyltransferase, is important in the regulation of the activity of these enzymes (53-55). Moreover, translocation of a macrophage PLAB has been observed in response to treatment of cells with diacylglycerol (56). Mouse mammary carcinoma-derived cells had a higher PLA2 activity than normal gland-derived cells with either arachidonoyl containing phosphatidylcholine or phosphatidylethanolamine as a substrate. This increased activity in tumor cell preparations may result in increased eicosanoid synthesis. The increased activity, with either substrate, in tumor versus normal cell preparations would suggest that there is one enzyme which can hydrolyze both substrates or that there is coordinate regulation of activity of two enzymes. The increased tumor cell PLA2 activity may be due to an increased amount of enzyme, increased specific activity of the enzyme, e.g., due to changes in post-translational modifications, or alterations in inhibitory or stimulatory factors. Stimulation of PLA2 activity, in other cell systems, was observed in response to a range of factors including components of signal transduction pathways, such as diacylglycerol and G proteins (57-62). Moreover, another GTP binding protein, the ras oncogene product, was associated with elevated PLA2 activity (16, 17, 63). The combined studies suggest the intriguing possibility that alterations in PLA2 activity may be a feature of a range of neoplastic systems and may relate to alterations in signal transduction systems in neoplasia. ACKNOWLEDGMENTS This work was supported by Grant CA25730 and Biomedical Research Support Grant RR05374 from the National Institutes of Health. The excellent technical assistance of Teresa Perrone is gratefully acknowledged. Dr. Jesse Sisken and Terri Newcombe are thanked for the fura2 analyses.
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