From the Centre de Recherche en Rhumatologie et
Immunologie, Centre de Recherche du Centre Hospitalier Universitaire de
Québec, Pavillon CHUL and Faculté de Médecine,
Université Laval, Québec G1V 4G2, Canada and
§ Pilot Therapeutics Inc.,
Winston-Salem, North Carolina 27101
Received for publication, July 12, 2000, and in revised form, February 21, 2001
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ABSTRACT |
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In this study
arachidonate-phospholipid remodeling was investigated in resting and
proliferating human T lymphocytes. Lymphocytes induced to proliferate
with either the mitogen concanavalin A or with anti-CD3 (OKT3) in
combination with interleukin 2 (OKT3/IL-2) showed a greatly accelerated
rate of [3H]arachidonate-phospholipid remodeling
compared with resting lymphocytes or with lymphocytes stimulated with
OKT3 or IL-2 alone. The concanavalin A-stimulated cells showed a 2-fold
increase in the specific activity of CoA-independent transacylase
compared with unstimulated cells, indicating that this enzyme is
inducible. Stimulation with OKT3 resulted in greatly increased
quantities of the group VI calcium-independent phospholipase
A2 but not of the quantity of group IV cytosolic phospholipase A2. However, group IV phospholipase
A2 became phosphorylated in OKT3-stimulated cells, as
determined by decreased electrophoretic mobility. Incubation of cells
with the group VI phospholipase A2 inhibitor, bromoenol
lactone, or the dual group IV/group VI phospholipase A2
inhibitor, methyl arachidonyl fluorophosphonate, did not block
arachidonate-phospholipid remodeling resting or proliferating T cells,
suggesting that these phospholipases A2 were not involved
in arachidonate-phospholipid remodeling. The incubation of
nonproliferating human lymphocytes with inhibitors of CoA-independent
transacylase had little impact on cell survival. In contrast,
OKT3/IL-2-stimulated T lymphocytes were very sensitive to apoptosis
induced by CoA-independent transacylase inhibitors. Altogether these
results indicate that increased arachidonate-phospholipid remodeling is
associated with T cell proliferation and that CoA-independent transacylase may be a novel therapeutic target for proliferative disorders.
Much effort has been expended to understand the mechanisms
by which inflammatory cells regulate the release of arachidonic acid
(AA)1 from cellular
phospholipids and the subsequent synthesis of AA-derived inflammatory
metabolites including the prostaglandins and leukotrienes. In addition
to regulating the activation of phospholipase(s) A2 (PLA2), which catalyzes the release of AA from
phospholipids, it has been shown that inflammatory cells also regulate
the distribution of this polyunsaturated fatty acid in cellular
phospholipids via an arachidonate-phospholipid (PL)-remodeling pathway
(1-3) (Fig. 1). This remodeling pathway
involves the sequential movement of arachidonate from
1-acyl-2-arachidonylglycerophosphocholine species to
1-alkyl-2-arachidonylglycerophosphocholine,
1-acyl-2-arachidonylglycerophosphoethanolamine, and
1-alk-1-enyl-2-arachidonylglycerophosphoethanolamine species. The
arachidonate-PL remodeling pathway is distinct from other phospholipid-remodeling activities such as base exchange (4) or
phosphatidylethanolamine N-methyltransferase-driven
remodeling (5) since the CoA-independent transacylase
(CoA-IT)-catalyzed step is specific for long chain polyunsaturated
fatty acids like arachidonic acid; phospholipids containing saturated,
monounsaturated, or even di-unsaturated fatty acids like linoleic acid
in the sn-2 position of the glycerol moiety do not show this
characteristic remodeling pattern (6) and are not utilized as
substrates by CoA-IT (7, 8).
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
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Fig. 1.
Schematic representation of
arachidonate-phospholipid remodeling between glycerophosphocholine
(GPC) and glycerophosphoethanolamine
(GPE) species.
Arachidonate-phospholipid remodeling is largely characterized by the CoA-IT-driven transfer of arachidonate between phospholipid species and a PLA2 catalyzed release of free arachidonic acid (1, 7, 9, 10). Although the calcium-independent group VI PLA2 (iPLA2) has been suggested to be associated with arachidonate-PL remodeling (11), the PLA2 isotype associated with this pathway has not been definitively identified.
The rate of arachidonate-PL remodeling is transiently enhanced when leukocytes like neutrophils or mast cells are stimulated with agonists (12, 13). This increased flux of arachidonate between phospholipid species has been hypothesized to be involved in regulating the release of AA by moving arachidonate into phospholipid pools that are accessible to arachidonate-specific PLA2(s). In an effort to uncover molecules that selectively inhibit the release of arachidonic acid, structurally different classes of compounds were discovered that inhibit CoA-IT (14, 15). These compounds were shown to be effective inhibitors of arachidonic acid release and, consequently, of leukotriene synthesis in activated leukocytes (16). It was recently observed that several cell lines including human promyelocytic HL-60 cells, monocyte-like THP-1 cells, macrophage-like P388D1 cells, and the breast cancer cell lines MCF-7 and MDA-MB-231 remodel AA very rapidly through the arachidonate-PL pathway (17-19). When these neoplastic cell lines are incubated with CoA-IT inhibitors, not only is arachidonate-PL remodeling effectively inhibited, but the cells readily undergo cell cycle arrest and apoptotic cell death (18, 20, 21). Importantly, isomers of CoA-IT inhibitors, which possess no inhibitory activity against CoA-IT, do not induce apoptosis in these cells (20). AA itself seems to play a central role in CoA-IT inhibitor-induced apoptosis since arachidonate-depleted HL-60 cells are much less susceptible to inhibitor-induced apoptosis than non-depleted cells (22). Since arachidonate is rapidly remodeled in these cell lines, CoA-IT inhibition leads to a marked accumulation of free AA, which may impart an anti-proliferative or pro-apoptotic signal through ceramide generation or after its transformation to oxygenated derivatives (18, 22). A similar mechanism of cell death triggered by an accumulation of free AA has been proposed for cells treated with cyclooxygenase inhibitors (23).
The susceptibility of these proliferating neoplastic cell lines to
apoptosis induced by CoA-IT inhibitors may be related to their rapid
rate of arachidonate-PL remodeling. However, it is not known if
enhanced remodeling activity is a characteristic of proliferating cells
or whether accelerated remodeling necessarily renders cells sensitive
to apoptosis after CoA-IT inhibition. In the present study, human
peripheral blood T lymphocytes were used to determine whether flux
through the arachidonate-PL-remodeling pathway is enhanced when
non-neoplastic cells are induced to proliferate. Our results show that
the rate of arachidonate-PL remodeling is greatly increased when human
T cells move out of G0 and begin to divide. This enhanced
rate of arachidonate-PL remodeling was associated with an induction of
CoA-IT activity but did not depend on iPLA2 or group IV
cytosolic PLA2 (cPLA2) activity. Importantly, proliferating but not resting T cells were very sensitive to cell death
induced by CoA-IT inhibitors. These results suggest that accelerated
arachidonate-PL remodeling is a characteristic of proliferating cells
and that CoA-IT may be a novel therapeutic target for proliferative disorders.
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MATERIALS AND METHODS |
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Reagents-- The [5, 6, 8, 9, 11, 12, 14, 15-3H]arachidonic acid (204 Ci/mmol) and 1-O-[3H]alkyl(octadecyl)-2-lyso-phosphatidylcholine (183 Ci/mmol) were purchased from Amersham Pharmacia Biotech. The [9,10-3H]oleic acid (14 Ci/mmol) was from Mandel-PerkinElmer Life Sciences. Arachidonic acid, 1-O-alkyl(octadecyl)-2-lyso-phosphatidylcholine (lyso platelet-activating factor), phosphatidylcholine (PC), phosphatidylethanolamine (PE), phosphatidylserine (PS), phosphatidylinositol (PI), phospholipase C from Bacillus cereus, and concanavalin A (ConA) from Canavalia ensiformis (Jack bean) were purchased from Sigma. RPMI 1640, fetal bovine serum, Ficoll type 400 (density of 1.077 g/ml), and Hanks' balanced salt solution were obtained from Wisent (Saint-Bruno, Québec, Canada). Percoll (density of 1.129 g/ml) was obtained from Amersham Pharmacia Biotech. Essential fatty acid free bovine serum albumin was purchased from ICN Biomedical Co. (Costa Mesa, Ca). TLC plates (20 × 20) and HPLC grade solvents were from Fisher. The silica HPLC column (250 × 4.60 mm, Spheroclone) was from Phenomenex (Torrance, Ca). The CoA-IT inhibitors SK&F 98625 (diethyl-7(3,4,5,-triphenyl-2-oxo-2,3-dihydroimidazol-1-yl)hepatin phosphate) and SK&F 45905 (2-[2-3,4-chloro-3-(trifluoromethyl)-phenyl)ureido]-4-(trifluoromethylphenoxy)-4,5-dichlorobenzene sulfonic acid) and SB 203347 (2-[2-[3,5-bis(trifluoromethyl) sulfamido]-4-trifluoromethylphenoxy] benzoic acid)) were generous gifts from Drs. James D. Winkler and Lisa Marshall, respectively (SmithKline Beecham Pharmaceuticals, King of Prussia, PA). LY 311727 (3-(3-acetamide-1-benzyl-2-ethylindolyl-5-oxy)propanephosphonic acid) was a generous gift from Dr. E. Michelich (Ely Lilly, Indianapolis, IN). Methyl arachidonylfluorophosphonate (MAFP) and the bromoenol lactone (BEL) were obtained from Cayman Chemical Co. (Ann Arbor, MI). The anti-CD3 was produced and purified from the OKT3 clone (a gift from Dr. Walid Mourad, Center Hospitalier de l'Université Laval, Québec, Canada).
Lymphocyte Preparation and Culture-- Human lymphocytes were isolated from the heparinized blood of healthy donors as previously described (24). Briefly, lymphocytes (>85% purity) were prepared by sequential centrifugation on Ficoll and Percoll gradients. In experiments where >99% pure lymphocytes were utilized, cells were further enriched by cell sorting after gating on lymphocytes on the basis of forward/side scatter (Epics Elite ESP, Coulter, Miami, FL). After their isolation, cells (1 × 106 cells/ml) were incubated for the indicated times at 37 °C in a 5% CO2 atmosphere with or without 10 µg/ml ConA or 1 µg/ml OKT3. In cells incubated with OKT3, 20 units/ml IL-2 were added 24 h after the addition of OKT3 unless indicated otherwise.
Pulse-labeling of Cells with [3H]Arachidonic Acid or [3H]Oleic Acid-- Cells that had been incubated for the indicated times were centrifuged and resuspended in 200 µl of 10% fetal bovine serum in RPMI. One hundred µl of incubation medium containing [3H]arachidonic acid or [3H]oleic acid (3 µCi/1 × 107 cells) were added to cell suspensions that were then incubated for 30 min at 37 °C in a shaking water bath. The cells were then washed twice with 10% fetal bovine serum in RPMI and resuspended in their original incubation medium. In some experiments, BEL, MAFP, SB 203347, or LY311727 were added to the cells immediately after pulse-labeling.
Extraction and Analysis of Lipids-- Incubations were terminated by washing cells once by centrifugation in cold Hanks' balanced salt solution followed by extraction of cellular lipids with organic solvents (25). The phospholipid classes of the extracts were separated by HPLC using a silica column with a hexane, 2-propanol, ethanol, 25 mM phosphate buffer (pH 7.4), acetic acid (490:367:100:30:0.6, v/v/v/v/v) mobile phase. After 5 min, the composition of phosphate buffer was increased from 3 to 5%, the acetic acid composition was decreased from 0.06 to 0.02% over a 5-min period, and these concentrations were maintained for the remainder of the run (26). Fractions containing neutral lipids, PE, PI/PS, and PC were collected. The extracts were dried under N2, and the [3H]arachidonate content of each fraction was measured by liquid scintillation counting (Beckman Instruments LS 5000 CE).
The phospholipid subclass analysis was performed as previously described (17). The PE and PC fractions isolated by HPLC were dried under a nitrogen stream and vortexed in 1 ml of 100 mM Tris buffer (pH 7.4). Phospholipase C, 40 and 20 units, was added to the PE and PC fractions, respectively, followed by 2 ml of ethyl ether, and the solutions were incubated for 6 h at 37 °C in a shaking water bath. The resulting diglycerides were extracted with 2 ml of hexane and then again with 2 ml of hexane/ether (1:1, v/v). The pooled extracts were dried under a stream of nitrogen, and acetylated derivatives were prepared by incubating overnight in pyridine/acetic anhydride (1:5, v/v) at 37 °C in a shaking water bath. The solution was dried under N2 and extracted twice with ether/hexane (1:1, v/v). The extract was washed once with 1 ml of water, and the subclasses were separated by TLC using a benzene/hexane/ether (50:45:4, v/v/v) mobile phase. The areas containing 1-acyl, 1-alkyl, and 1-alk-1-enyl-linked lipids were identified by co-migration of standards, which were visualized with iodine. The corresponding areas were scraped, and associated radioactivity was determined by liquid scintillation counting.
CoA-IT Activity Assay-- CoA-IT activity was measured in lymphocyte membrane preparations. Cells were first sonicated in 80 mM KCl, 10 mM Hepes, 1 mM EGTA, 1 mM EDTA (pH 7.4), 5 µg/ml leupeptin, 10 mM NaF, and 0.2 mM NaVO3. The sonicate was then centrifuged at 8000 × g for 15 min (4 °C), and the resulting supernatant was centrifuged at 100,000 × g for 45 min (4 °C). The resulting pellet was resuspended in PBS buffer (pH 7.4) containing EGTA (1 mM). The CoA-IT activity assay was performed as described previously (27) in a total volume of 100 µl containing 3 µg of membrane protein. The reaction was initiated by the addition of 1-O-[3H]alkyl(octadecyl)-2-lyso-PC (0.5 µCi/assay) diluted in assay buffer containing 250 µg/ml bovine serum albumin and a varied (0 to 1 µM) final concentration of unlabeled 1-O-alkyl(octadecyl)-2-lyso-PC. The reaction was allowed to proceed for 10 min at 37 °C and was stopped with 300 µl of chloroform/methanol (1:2 v/v). The lipids were then extracted (25), and lyso-PC and PC standards were added as carrier lipids. Lipids were separated by TLC using chloroform/methanol/acetic acid/water (50:25:8:4 v/v/v/v). For kinetic analysis experiments, the assay was configured so that less than 5% of the substrate was converted to product in order to ensure that zero order kinetics could be applied.
Determination of Apoptosis-- After the indicated times of cell culture, apoptosis was determined using the annexin V-PI apoptosis detection kit according to the manufacturer's instructions (R & D Systems, Minneapolis, MN) and by poly(ADP-ribose) polymerase (PARP) cleavage, detected by immunoblotting using the monoclonal antibody C2-10 directed against PARP (28) (a generous gift from Dr Guy Poirier, Center Hospitalier de l'Université Laval, Québec, Canada).
Immunoblot Analysis of iPLA2 and cPLA2-- Lymphocyte cytosolic proteins (50 µg) were separated by SDS-polyacrylamide gel electrophoresis (29) and transferred to a nitrocellulose membrane that was blocked 30 min at room temperature with 5% milk protein in TBS-Tween (25 mM Tris-HCl, 190 mM NaCl (pH 7.6), and 0.15% Tween 20). The membrane was then washed in TBS-Tween and incubated with polyclonal antibody to iPLA2 (Cayman Chemical Co, Ann Arbor, MI) (1:500 dilution v/v) or with a 1:500 dilution (v/v) of the polyclonal antibody to cPLA2, MF 142 (a generous gift from Dr. Philip Weech of Merck Frosst, Canada), in 1% milk protein in TBS-Tween for 1 h at room temperature. The nitrocellulose membranes were then washed 5 times for 5 min each in TBS-Tween and incubated for 1 h at room temperature with a horseradish peroxidase-linked goat anti-rabbit IgG (1:10 000) (Bio-Can Scientific, Mississauga, Ontario, Canada) diluted in TBS-Tween containing 1% milk protein. After having washed the membrane 5 times with TBS-Tween for 5 min each, the antibody complex was visualized by enhanced chemiluminescence Plus (Mandel-PerkinElmer Life Sciences).
[3H]Thymidine Incorporation-- Lymphocytes (1 × 10 6 cells/ml) incubated for the various incubation times were exposed to [3H]thymidine (1 µCi/well containing 200,000 cells) for 18 h. The incorporated radioactivity was collected on a glass fiber filter (Printed Filtermat A, Wallac Oy, Turku, Finland) with a cell harvester (Tomtec, Orange, Conn), and the radioactivity was quantified with a liquid scintillation counter 1450 Microbeta (Wallac Oy, Turku, Finland).
Protein Determination--
Protein content was determined
according to a modification of the Lowry method as previously described
(30).
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RESULTS |
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Lymphocyte Proliferation and Arachidonate-Phospholipid
Remodeling--
Rapidly proliferating transformed cell lines exhibit
very rapid arachidonate-PL remodeling (17-19). These observations
suggest that either cell transformation induces an increased flux of
arachidonate through this remodeling pathway or that this pathway is
accelerated when cells enter the cell cycle and proliferate. Therefore,
a first set of experiments investigated whether the induction of cell
proliferation results in an enhanced arachidonate-PL remodeling in
nontransformed cells. Human lymphocytes were isolated from the
peripheral blood of healthy volunteers and incubated without the
addition of a stimulus or stimulated with either the T cell mitogen
ConA, IL-2, the anti-CD3 antibody OKT3, or with OKT3 followed 24 h
later with IL-2 (OKT3/IL-2). When freshly isolated lymphocytes were
pulse-labeled with [3H]AA and allowed to incubate for up
to 24 h, a slow but measurable redistribution of the radiolabel
from PC to PE species was observed (Fig.
2A). This redistribution of
arachidonate from PC species to PE species is a hallmark of
arachidonate-phospholipid remodeling. A similarly slow but measurable
rate of [3H]arachidonate redistribution between
phospholipid species was observed when unstimulated cells were
pulse-labeled after 1, 2, 3, or 4 days of incubation (data not shown).
When freshly isolated, [3H]arachidonate-pulse-labeled
cells were stimulated with ConA for 24 h; a similarly slow
redistribution of the radiolabel from PC to PE species was also
observed. As expected, during this initial 24-h period little induction
of cell proliferation was observed in ConA-stimulated cells as
determined by [3H]thymidine incorporation and cell
counting (data not shown). However, when T cells were stimulated with
either ConA or OKT3/IL-2 for 3 days, cells underwent rapid
proliferation, as measured by [3H]thymidine incorporation
(Fig. 3) and cell counting (data not shown). These cells were determined to be >99% pure T cells after CD3
staining and fluorescence-activated cell sorter analysis (data not
shown). When these 3 day-stimulated T cells were then pulse-labeled with [3H]AA and allowed to incubate further, a very rapid
redistribution of [3H]arachidonate from PC to PE species
was observed (Fig. 2, B and C). Stimulation of T
cells with either OKT3 or IL-2 alone did not induce the degree of cell
proliferation (Fig. 3) or increased rate of remodeling (Fig. 2,
D and E) observed in ConA- or
OKT3/IL-2-stimulated cells; however, treatment of cells with OKT3 alone
did result in an induction of cell proliferation that was associated
with a more rapid rate of arachidonate-PL remodeling than that observed in resting cells.
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The very rapid redistribution of [3H]arachidonate from PC to PE species observed in cells stimulated with ConA or with OKT3/IL-2 was characteristic of arachidonate-phospholipid remodeling since 1) the movement of [3H]arachidonate was mainly from 1-acyl-linked PC species to 1-alkyl-, 1-alk-1-enyl-, and 1-acyl-linked-PE species (Table I), 2) no net redistribution of [3H]arachidonate to or from PI/PS species was observed because the amount of [3H]arachidonate in these phospholipid classes remained constant during the incubation period (Fig. 2, B and C), and 3) cells pulse-labeled with [3H]oleate showed no redistribution of radiolabel between different phospholipid classes (Table I). Altogether, these results indicate that a very rapid rate of arachidonate-PL remodeling is associated with the proliferation of peripheral blood T cells.
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CoA-IT Activity--
The remodeling of arachidonate between
phospholipid species requires several enzymatic activities. The step
that is unique to this pathway is that which is catalyzed by CoA-IT.
This enzyme has not yet been isolated; however, its activity has been
characterized and can be assayed in broken cell preparations (27). To
determine whether the enhanced remodeling measured in proliferating T
cells may be associated with an increase in CoA-IT activity,
100,000 × g particulate fractions were prepared from
both unstimulated and ConA-stimulated lymphocytes, and CoA-IT activity
was assayed. As can be seen in Fig. 4,
100,000 × g fractions from stimulated cells
consistently showed an ~2-fold increase in CoA-IT activity compared
with unstimulated cells, indicating that the enhanced flux of
arachidonate through the remodeling pathway observed in stimulated
cells was associated with an induction of CoA-IT activity.
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Phospholipase(s) A2 in Lymphocytes-- Another key step associated with this remodeling pathway is that catalyzed by a PLA2 (Fig. 1). However, the PLA2 isotype involved in arachidonate-PL remodeling has not been definitively identified. It was previously suggested that iPLA2 may be involved in arachidonate-PL remodeling because the treatment of P388D1 cells with the iPLA2 inhibitor, BEL, inhibits the incorporation of AA into cellular phospholipids (11). However, since arachidonate-PL remodeling is specific to highly unsaturated fatty acids like AA (6, 10), an arachidonate-specific PLA2 may also be involved in generating the lysophospholipid species used as acceptor phospholipids in the CoA-IT-catalyzed reaction. For that reason, the arachidonate-specific group IV cPLA2 could be a candidate for this step in the pathway.
Therefore, the expression of these two PLA2 isotypes was
determined in resting and stimulated T cells. Fig.
5 shows that although resting T cells
expressed cPLA2, very little if any iPLA2 was detected by immunoblot analysis. However, when T cells were stimulated with OKT3/IL-2, iPLA2 protein levels were greatly
increased. Although co-stimulation with anti-CD3 and IL-2 was required
for enhanced arachidonate-PL remodeling, CD3 stimulation alone was
sufficient to induce iPLA2 expression. Although no apparent
increase in the protein content of cPLA2 was measured in
stimulated cells, the cPLA2 from CD3-stimulated T cells
migrated more slowly on polyacrylamide gels than that from unstimulated
cells. Identical results were obtained in lymphocytes purified by cell
sorting and stimulated with ConA (data not shown). This gel shift of
cPLA2 has been shown to be due to the phosphorylation of
the protein on serine residue 505 and is associated with the activation
of the enzyme (31). As with iPLA2 expression, CD3
stimulation alone in the absence of added IL-2 was sufficient to induce
cPLA2 phosphorylation in human T cells.
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PLA2 Inhibition Studies-- The up-regulation of iPLA2 and phosphorylation of cPLA2 after cell stimulation suggested that these PLA2 isotypes may be associated with the enhanced arachidonate-PL remodeling described above. Therefore, the impact of different PLA2 inhibitors on the remodeling process was evaluated. T cells were stimulated with ConA or OKT3/IL-2 for 72 h and then pulse-labeled with [3H]AA and incubated in the presence of the irreversible iPLA2 inhibitor BEL (32) or the irreversible dual iPLA2-cPLA2 inhibitor MAFP (33, 34). As can be seen in Table II, the incubation of cells for 5 h with 12.5 µM BEL, a concentration shown to inhibit AA incorporation in P388D1 cells (11), had no effect on the sequential movement of [3H]arachidonate between phospholipid species even though the IC50 of this irreversible iPLA2 inhibitor is 60 nM (32). More prolonged incubation (15 h) of either resting or stimulated T cells with 12.5 µM BEL was cytotoxic to these cells. Similarly, higher concentrations of BEL (25 µM) was also cytotoxic to both resting and stimulated cells, inducing rapid and complete cell death within 5 h (data not shown). BEL is also known to inhibit phosphatidate phosphohydrolase (35, 36). When cells were incubated with another phosphatidate phosphohydrolase inhibitor, propranolol (100 µM), cell death was also readily induced, as determined by annexin V-PI staining and PARP cleavage (data not shown), suggesting that this enzyme may be required for T cell survival.
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When stimulated cells were incubated with the irreversible dual iPLA2/cPLA2 inhibitor MAFP, no effect on arachidonate-PL remodeling was observed when concentrations as high as 50 µM were added to the incubation medium for up to 15 h (Table II). MAFP inhibits both iPLA2 and cPLA2 in the high nM range (33, 37, 38), and in whole cells MAFP has been shown to inhibit the release of AA from stimulated human neutrophils and platelets with an IC50 in the range of 10 nM-1 µM (38-40). These results indicate that although mitogen stimulation of human T cells results in an up-regulation of the group VI iPLA2 and the phosphorylation of the arachidonate-specific group IV cPLA2, another as yet undetermined PLA2 isotype that is not inhibited by MAFP or BEL may be involved in arachidonate-PL remodeling in human T cells.
Inhibitors of CoA-IT Induce Apoptosis in Proliferating but Not
Resting T Cells--
The sensitivity of transformed cell lines to
CoA-IT inhibitors has been attributed to the very rapid rate of
arachidonate-PL remodeling in these cells (22). We hypothesized that
cells that slowly remodel arachidonate may be less sensitive to CoA-IT
inhibitor-induced apoptosis but that an increased rate of
arachidonate-PL remodeling would render cells sensitive to CoA-IT
inhibition. The observation that proliferating T cells exhibited
greatly accelerated arachidonate-PL remodeling compared with resting
cells provided an excellent model to test this hypothesis. When
resting, nonstimulated lymphocytes were incubated with CoA-IT
inhibitors; no induction of apoptosis was measured as assessed by PARP
cleavage (Fig. 6). However, T cells
stimulated with OKT3/IL-2 were very sensitive to the induction of cell
death by the CoA-IT inhibitor, SK&F 98625, resulting in a complete
cleavage of PARP in these cells (Fig. 6). Cleavage of PARP is an
indication of caspase 3-like activity since PARP is characteristically
cleaved at a DEVD site, resulting in the disappearance of the 116-kDa
parent protein and the specific appearance of the 89-kDa cleavage
product. Interestingly, 10 times more protein from nonstimulated cells
than from stimulated cells was loaded onto the gels, indicating that
PARP protein levels were increased in stimulated cells.
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The dose response to CoA-IT inhibitors was assessed using the annexin
V-PI-staining method, which measures the loss of membrane asymetry
associated with apoptosis and which allows a more quantitative assessment of cell death. As can be seen in Fig.
7A, resting cells or cells
stimulated with IL-2 alone underwent little apoptosis when incubated
with up to 30 µM of the CoA-IT inhibitor SK&F 98625. This
concentration was previously shown to be very effective in inducing
apoptosis in HL-60 cells (20). On the other hand, T cells co-stimulated
with OKT3/IL-2 that exhibit rapid arachidonate-PL remodeling were much
more sensitive to CoA-IT inhibitors and underwent apoptosis in a
dose-dependent manner when incubated with SK&F 98625. Interestingly, cells stimulated with OKT3 showed an intermediate sensitivity to CoA-IT inhibition, which corresponded to the sub-optimal induction of cell proliferation and arachidonate-PL remodeling measured
in these cells. This suggests that the sensitivity of T cells to
apoptosis induced by CoA-IT inhibitors is linked to their rate of
arachidonate-PL remodeling.
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The pattern of annexin V and PI staining in resting and
OKT3/IL-2-stimulated cells is shown in Fig. 7B. The annexin
V staining and PI uptake obtained after the incubation of these cells
with CoA-IT inhibitors shows a typical apoptotic pattern with the
progression of double negative cells toward double positive cells with
an annexin V-positive/PI-negative intermediate. Although this sensitive method measured a small increase in annexin V-positive cells when resting cells were incubated with CoA-IT inhibitors, both OKT3/IL-2 and
ConA (not shown)-stimulated T cells were much more responsive to the
induction of apoptosis after incubation with SK&F 98625 (Fig.
7B) or the structurally distinct CoA-IT inhibitor, SK&F 45905 (data not shown). Overall, these results are consistent with the
hypothesis that cells in which the arachidonate-PL-remodeling pathway
is accelerated become sensitive to the induction of apoptosis as a
result of CoA-IT inhibition.
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DISCUSSION |
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Previous studies show that arachidonate-PL remodeling occurs at a very rapid rate in neoplastic cell lines (19, 21, 22) and that these cells undergo cell cycle arrest and apoptosis when treated with inhibitors of CoA-IT (18, 20, 21). The hypothesis in the present study was that arachidonate-PL remodeling would be accelerated when non-neoplastic cells are induced to proliferate and that such cells would be rendered sensitive to the induction of apoptosis by CoA-IT inhibitors. The results presented herein support this hypothesis by showing that mitogen-stimulated human T cells exhibit very rapid arachidonate-PL remodeling once they begin to proliferate compared with resting cells. Importantly, these proliferating T cells also become sensitive to the action of CoA-IT inhibitors, which effectively induce apoptotic cell death in stimulated but not resting human T cells.
Human peripheral blood T cells were an ideal model in which to compare arachidonate-PL remodeling in resting and proliferating cells since they readily and reproducibly enter the cell cycle upon appropriate stimulation. The results presented here clearly indicate that when human T cells are stimulated with a mitogen or with activating anti-CD3 antibodies in the presence of IL-2, the induction of cell proliferation is accompanied by an acceleration of the arachidonate-phospholipid-remodeling pathway. In addition to supporting the premise that an increased rate of remodeling is a characteristic of proliferating cells, T cells also provided a good model to study the regulation of arachidonate-PL remodeling. This pathway is characterized by the CoA-IT-driven transfer of arachidonate between phospholipid species but not that of shorter chain or more saturated fatty acids. This membrane-bound enzyme has not yet been isolated due to the difficulties in retaining enzymatic activity when particulate cell fractions are treated with detergents. Nonetheless, in mitogen-stimulated cells, the specific activity of CoA-IT was doubled compared with that of resting lymphocytes, indicating that this enzymatic activity is inducible. However, the flux of arachidonate between phospholipid species was increased severalfold since stimulated cells pulse-labeled with [3H]AA approached isotopic equilibrium within 5 h, whereas resting cells had not yet attained isotopic equilibrium after a 48-h incubation (data not shown). This suggests that other potential sites of regulation of this pathway may also be operative.
Phospholipase(s) A2 is usually involved in the regulation of cellular arachidonic acid release in acutely stimulated cells and may, therefore, also play a role in the regulation of arachidonate-PL remodeling. It has been suggested that the calcium-independent group VI iPLA2 may be the PLA2 isotype involved in the release of AA in the arachidonate-PL-remodeling pathway (11). The observation in the present study that this protein is up-regulated in CD3-stimulated T cells is consistent with its potential role in arachidonate-PL remodeling. However, the treatment of cells with the iPLA2 inhibitor BEL was without effect on the redistribution of radiolabeled arachidonate in pulse-labeled cells. This lack of effect of iPLA2 inhibition on arachidonate remodeling was confirmed in cells incubated with the dual iPLA2/cPLA2 inhibitor, MAFP, which did not alter the redistribution of radiolabeled AA in pulse-labeled cells at concentrations as high as 50 µM. Therefore, although the group VI iPLA2 is up-regulated, two different iPLA2 inhibitors used at concentrations that have been shown to inhibit AA release and iPLA2 activity in whole cells failed to affect the remodeling pathway. This result is in accord with a recent report in pancreatic beta cells where BEL had no effect on the movement of arachidonate from PC to PE species (36).
These inhibitor studies also suggest that the 85-kDa group IV cPLA2 is not involved in arachidonate-phospholipid remodeling. The arachidonate-specific cPLA2 was an obvious candidate to be involved in arachidonate-PL remodeling since it is the only PLA2 isotype described to date that shows high specificity for 2-arachidonyl-phospholipids (41, 42). The lack of effect of MAFP on remodeling is consistent with an earlier report that showed that cPLA2 is not involved in the CoA-IT-dependent generation of platelet-activating factor in ionophore-stimulated neutrophils (43). Interestingly, in the present study cPLA2 was activated in stimulated T cells since the characteristic gel shift that is associated with its phosphorylation and activation was observed. It is possible that cPLA2 activation plays a role in the previously described arachidonic acid release coupled to 5-lipoxygenase product synthesis in CD28-stimulated T cells (44). There is some evidence that secretory PLA2(s) like the group V or group IIa sPLA2 may in some cases have specificity for 2-arachidonyl-phospholipids or even a preference for phosphatidylethanolamine species over phosphatidylcholine species in certain conditions (45). However, no evidence for an increase in PLA2 activity in the incubation medium of stimulated cells could be demonstrated, and two separate sPLA2 inhibitors, SB 203347 and LY 311727, had no effect on remodeling (data not shown). Overall, these results cast doubt on the role of the known PLA2 isotypes most likely to be involved in arachidonate-PL remodeling and suggest that another, perhaps as yet unidentified, PLA2 may be involved in the arachidonate-PL-remodeling pathway.
Previous studies show that neoplastic cells lines undergo apoptosis when incubated with CoA-IT inhibitors (18, 20, 21). That these cell lines remodel arachidonate very rapidly suggested that their sensitivity to CoA-IT inhibitors may be related to the flux of arachidonate through this pathway. Having established in the present study that the arachidonate-PL-remodeling pathway was greatly accelerated after the induction of T cell proliferation, the sensitivity to CoA-IT inhibitors could be compared in cells that rapidly or slowly remodel arachidonate. The sensitivity of T cells to apoptosis induced by CoA-IT inhibition was very much dependent on whether the cells were rapidly undergoing arachidonate-PL remodeling. Unstimulated T cells were much less sensitive to the induction of apoptosis after CoA-IT inhibition compared with stimulated proliferating T cells. In fact, no cleavage of PARP was detected when resting cells were incubated with CoA-IT inhibitors. Consistent with the link between accelerated arachidonate-PL remodeling and sensitivity to CoA-IT inhibition, T cells that were stimulated for 24 h but that had not yet begun to proliferate or accelerate the remodeling pathway were also much less sensitive to CoA-IT inhibitors than proliferating T cells. The sensitivity of HL-60 cells, which rapidly remodel arachidonate, to CoA-IT inhibition was suggested to be due to the rapid accumulation of the pathway intermediate free AA and its activation of a sphingomyelinase leading to ceramide generation (22). A similar cell death mechanism where the cell's inability to control free AA levels leads to increased ceramide concentration has been suggested to occur after cPLA2 activation in TNF-sensitive cells (46, 47) and in cells treated with cyclooxygenase inhibitors (23). In resting cells where arachidonate-PL remodeling proceeds slowly, pathway intermediates like free AA would accumulate to a lesser extent after CoA-IT inhibition, likely allowing the cell to cope with any released free AA via reacylation into phospholipids or triglycerides.
The present study indicates that rapid arachidonate-PL remodeling is a
likely characteristic of proliferating cells and responsible for their
susceptibility to cell death resulting from CoA-IT inhibition. These
cells may require a rapid and specific organization of
arachidonyl-phospholipids to ensure that this polyunsaturated fatty
acid is properly distributed in the membrane phospholipids of newly
synthesized cell structures. Indeed, arachidonic acid metabolites are
involved in the regulation of numerous cellular processes, which would
explain why cells have developed a specific pathway to control its
distribution in phospholipid species. Importantly, the observation that
the induction of this remodeling pathway is associated with cell
proliferation and renders cells susceptible to apoptotic cell death
after the inhibition of CoA-IT supports the concept that CoA-IT may be
a specific therapeutic target for proliferative disorders including lymphoproliferation associated with auto-immune diseases or graft rejection.
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FOOTNOTES |
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* These studies were supported by a grant from the Medical Research Council of Canada and were presented in part at the 6th International Conference on Eicosanoids and Other Bioactive Lipids in Cancer, Inflammation-related Diseases, Boston, MA, September 12-15, 1999.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
¶ Recipient of a Scholarship from Le Fonds de la Recherche en Santé du Québec. To whom correspondence should be addressed: Pilot Therapeutics Inc., 101 N. Chestnut St., Winston-Salem, NC 27103. Tel.: 336-725-2222; Fax: 336-725-2221; E-mail: marc.surette@crchul.ulaval.ca.
Published, JBC Papers in Press, February 22, 2001, DOI 10.1074/jbc.M006152200
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ABBREVIATIONS |
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The abbreviations used are: AA, arachidonic acid; BEL, bromoenol lactone; CoA-IT, CoA-independent transacylase; PLA2, phospholipase A2; cytosolic PLA2, iPLA2, calcium-independent PLA2; HPLC, high performance liquid chromatography; MAFP, methyl arachidonyl fluorophosphonate; PARP, poly(ADP-ribose) polymerase; PC, phosphatidylcholine; PE, phosphatidylethanolamine; PI, phosphatidylinositol; PS, phosphatidylserine; PL, phospholipid; ConA, concanavalin A; TBS, Tris-buffered saline; IL-2, interleukin 2.
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