Perturbations in the control of cellular arachidonic acid levels block cell growth and induce apoptosis in HL-60 cells

Marc E. Surette1,4, Alfred N. Fonteh2, Chantale Bernatchez1 and Floyd H. Chilton2,3

1 Université Laval and Centre de Recherche en Rhumatologie et Immunologie, Centre Hospitalier Universitaire de Québec, Pavillon CHUL, 2705 Laurier, Ste-Foy, Québec G1V 4G2, Canada,
2 Section on Pulmonary and Critical Care Medicine and
3 Department of Biochemistry, Wake Forest University School of Medicine, Medical Center Boulevard, Winston-Salem, NC 27157–1054, USA


    Abstract
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Our previous studies demonstrated that inhibitors of arachidonate–phospholipid remodeling [i.e. the enzyme CoA-independent transacylase (CoA-IT)] decrease cell proliferation and induce apoptosis in neoplastic cells. The goal of the current study was to elucidate the molecular events associated with arachidonate–phospholipid remodeling that influence cell proliferation and survival. Initial experiments revealed the essential nature of cellular arachidonate to the signaling process by demonstrating that HL-60 cells depleted of arachidonate were more resistant to apoptosis induced by CoA-IT inhibition. In cells treated with CoA-IT inhibitors a marked increase in free arachidonic acid and AA-containing triglycerides were measured. TG enrichment was likely due to acylation of arachidonic acid into diglycerides and triglycerides via de novo glycerolipid biosynthesis. To determine the potential of free fatty acids to affect cell proliferation, HL-60 cells were incubated with varying concentrations of free fatty acids; exogenously provided 20-carbon polyunsaturated fatty acids caused a dose-dependent inhibition of cell proliferation, whereas oleic acid was without effect. Blocking 5-lipoxygenase or cyclooxygenases had no effect on the inhibition of cell proliferation induced by arachidonic acid or CoA-IT inhibitors. An increase in cell-associated ceramides (mainly in the 16:0-ceramide fraction) was measured in cells exposed to free arachidonic acid or to CoA-IT inhibitors. This study, in conjunction with other recent studies, suggests that perturbations in the control of cellular arachidonic acid levels affect cell proliferation and survival.

Abbreviations: AA, arachidonic acid; CoA-IT, CoA-independent transacylase; DG, diacylglycerol; ESI-MS/MS, electrospray ionization-tandem mass spectrometry; EtBr, ethidium bromide; FBS, fetal bovine serum; FSC, forward scatter; 2H3-SA, trideuterated stearic acid; 2H8-AA, octadeuterated AA; HAS, human serum albumin; HBSS, Hank's balanced salt solution; MG, monoacylglycerol; NICI-GC/MS, negative ion chemical ionization gas chromatography/mass spectrometry; TCA, trichloroacetic acid; TG, triacylglycerol; TLC, thin-layer chromatography.


    Introduction
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Apoptosis is an intrinsic cell suicide program that is morphologically distinct from necrotic cell death which results from overwhelming cell injury. This process plays an essential role in maintaining a balance with mitosis in the regulation of the number of animal cells and in mediating pathological processes associated with proliferative disorders such as cancer. Agents that induce apoptosis include physiological activators, such as the TNF family of ligands (TNF and Fas ligand), certain growth factors, some neurotransmitters and damage-related inducers, such as heat shock, radiation and viral infections (1). Although early signaling processes may differ, various inducers of apoptosis are likely to activate a common signaling pathway(s), which is not, as yet, fully defined in mammalian cells. Recently, the turnover of lipids has been implicated as playing central roles in the induction of apoptosis. The two pathways which have received the most attention in this regard are those involving sphingomyelin and arachidonic acid (AA) metabolism (28).

We have recently demonstrated that compounds which specifically inhibit an enzyme that controls the movement of arachidonate between phospholipid molecular species, CoA-independent transacylase (CoA-IT), blocks cell proliferation and induces apoptosis in HL-60 cells (9,10). In contrast, close structural analogs of these inhibitors which possess no inhibitory activity toward CoA-IT do not induce apoptosis. CoA-IT catalyses the transfer of arachidonate from 1-acyl-linked phospholipid pools, such as 1-acyl-2-arachidonoyl-sn-glycero-3-phosphocholine to 1-ether-linked phospholipids, such as 1-alk-1-enyl-2-arachidonoyl-sn-glycero-3-phosphoethanolamine (11,12). Consequently, the inhibition of CoA-IT leads to a preferential loss of arachidonate from 1-ether-linked phospholipid pools (9). The relationship between the inhibition of proliferation and arachidonate depletion from 1-ether-linked phospholipids is noteworthy since neoplastic cells have been recognized to have a high content of 1-ether linked phospholipids when compared with their non-malignant counterparts (1315).

Although the studies mentioned above indicate that the movement of arachidonate is critical to maintaining the capacity of cells to survive and proliferate, they also raise the fundamental question of how blocking the remodeling pathway generates a signal that stops cell proliferation and induces apoptosis. The current study reveals that the remodeling of arachidonate, orchestrated by CoA-IT, is necessary for cancer cells to maintain low levels of free unesterified AA. Moreover, when remodeling is blocked, the ensuing increase in free AA levels appears to be an important cellular signal for inhibiting cell proliferation and inducing apoptosis.


    Materials and methods
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Materials
Lipid standards triacylglycerol (TG), diacylglycerol (DG) and monoacylglycerol (MG) were obtained from Avanti Polar Lipids (Birmingham, AL). Fatty acid standards and ceramide standards were obtained from Cayman Chemical (Ann Arbor, MI). Essentially fatty acid-free human serum albumin (HSA), fetal bovine serum (FBS), penicillin–streptomycin, insulin–transferrin–sodium selenite mixture and common laboratory chemicals were obtained from Sigma (St Louis, MO). RPMI 1640 culture media and Hank's balanced salt solution (HBSS) were obtained from Gibco (Grand Island, NY). [5,6,8,9,12,14,15-3H]arachidonic acid was obtained from American Radiolabelled Chemicals (St Louis, MO). Uniplate Silica Gel G thin-layer chromatography (TLC) plates were obtained from Analtech Inc. (Newark, DE). Octadeuterated AA (2H8-AA) and trideuterated stearic acid (2H3-SA) were purchased from Biomol Research Laboratories (Plymouth Meeting, MA). Pentafluorobenzyl bromide (20% in acetonitrile) and di-isopropanolamine (20% in acetonitrile) were purchased from Pierce (Rockford, IL). SK&F 98625 [diethyl 7-(3,4,5-triphenyl-2-oxo-2,3-dihydroimidazol-1-yl)hepatine phosphonate] and SK&F 45905 (2-[2-(3,4-chloro-3-(trifluoromethyl)phenyl)ureido]-4-(trifluoromethylphenoxy)-4,5-dichlorobenzene sulfonic acid) (16) were kindly provided by Dr James D.Winkler (Smithkline Beecham, King of Prussia, PA). All solvents (HPLC grade) were purchased from Fisher Scientific (Silver Spring, MD).

Cells
The human promyelocytic cell line HL-60 was obtained from the American Type Culture Collection (Rockville, MD). Cells were maintained in RPMI media containing penicillin (250 U/ml) and streptomycin (250 µg/ml), and supplemented with 10% FBS.

Experiments utilizing cells grown in serum-free media
In some experiments, cells were maintained for at least 10 passages in RPMI media containing penicillin (250 U/ml), streptomycin (250 µg/ml), insulin (10 µg/ml), transferrin (10 µg/ml), sodium selenite (10 ng/ml), BSA (0.1%) and L-glutamine (4 mM) (17). In these experiments, cells grown in serum-free media in the presence or absence of 3 µM arachidonic acid were incubated with the indicated concentrations of CoA-IT inhibitors. Cell death was assessed in these cells by flow cytometry as described below.

Extraction and analysis of lipids
Cells were removed from culture and washed (x2) with ice-cold HBSS. Cellular lipids were then extracted by the method of Bligh and Dyer (18). For the determination of cellular arachidonate content, a fraction of the lipid extract containing [2H]8-AA and [2H]3-SA as internal standards was submitted to methanolic base hydrolysis by heating in methanol:water (75:25 v/v) containing 2 N KOH at 60°C for 30 min. The solution was diluted with 8 vol of water, acidified with 1 vol of 6 N HCl and the lipids were extracted by loading the solution onto octadecyl columns. The columns were washed with 2 ml of water and the fatty acids were then eluted from the column with 4 ml of methanol. Pentafluorobenzylester derivatives of fatty acids were prepared and the quantities of fatty acids were determined by negative ion chemical ionization gas chromatography/mass spectrometry (NICI-GC/MS), as previously described (19).

Cellular ceramides were quantitated using a previously described method (20) with some modifications as described below. Briefly, cell lipid extracts containing C6-ceramide as an internal standard were dried under a stream of nitrogen to evaporate the solvent, were solubilized in 20 µl chloroform and were diluted to a final volume of 100 µl with a solution of 5 mM ammonium acetate in glass-distilled ethanol. Samples were then analysed by electrospray ionization-tandem mass spectrometry (ESI-MS/MS) using a nebulizer-assisted electrospray (ion spray) interface coupled to a triple-quadrupole MS (API-III; PE Sciex, Thornhill, Ontario, Canada). Samples were infused into the electrospray source at rate of 10 µl/min. Positive ion MS/MS used an orifice voltage of 65 V. The third quadrupole was selected for a fixed daughter ion of m/z 264 and the first quadrupole was scanned for parent ions with a mass step of 0.5 Da and a rate of 2 ms/step. A total of 50 scans were collected over a mass range of 350–675 Da.

Labeling of cellular glycerolipids to isotopic equilibrium with [3H]arachidonic acid
In experiments where cellular glycerolipids were labeled to isotopic equilibrium with [3H]AA (200 Ci/mmol), cells were removed from media by centrifugation (200 g, 10 min, 4°C) and resuspended at 20x106 cells/ml in Ca2+-free HBSS. [3H]AA (2 µCi/20x106 cells) was added in 200 µl of HBSS containing HSA (250 µg/ml) and the cells were incubated for 30 min at 37°C. The cells were then washed once by centrifugation (200 g, 10 min, 4°C) with HBSS containing 250 µg HSA/ml, resuspended in culture media (2x105 cells/ml) and allowed to incubate for 48 h at 37°C. After 48 h, cells were washed followed by centrifugation (200 g, 10 min, 4°C) and then placed in culture media at a concentration of 5x105 cells/ml. CoA-IT inhibitors [SK&F 45905 (50 µM) or SK&F 98625 (25 µM)] were then added to the cell suspension. After the indicated times of incubation at 37°C, aliquots of the cell suspension were removed, a small fraction was measured for total radioactivity by liquid scintillation spectroscopy, the remainder of the cell suspension was then centrifuged (200 g, 10 min, 4°C) and the cell-free supernatant fluid was separated from the cell pellet. The radioactivity in an aliquot of the supernatant fluid was determined and the supernatant lipids were extracted as described above. The cell pellet was washed with HBSS containing 250 µg/ml HSA and the cellular lipids were extracted as described above. Neutral lipids from the extracts were separated by TLC eluted with hexane:ether:formic acid (90:60:6 v/v). Radioactive products were visualized by radioscanning (BioScan Scanner, Washington, DC) and their migration compared with that of authentic standards (phospholipids, MG, DG, TG and free AA). The products were then scraped and radioactivity determined by liquid scintillation spectroscopy.

Metabolism of exogenous fatty acids
HL-60 cells were suspended in culture media (5x105 cells/ml) that was supplemented with 30 µM [3H]AA (30 Ci/mol). The cells were incubated at 37°C and at the indicated period of time aliquots were removed, the cells were washed twice in ice-cold HBSS containing 250 µg/ml HSA and the cellular lipids were extracted as described above. Materials from the extraction were separated by TLC, the products were scraped and radioactivity determined by liquid scintillation spectroscopy as described above.

Cell proliferation assays
Cells (2x105/ml) were incubated in growth media with various concentrations of fatty acids or CoA-IT inhibitors as described in the figure legends. After 24 h, 200 µl aliquots of cells were incubated in triplicate on 96-well plates in the presence of [3H]thymidine (0.5 µCi/well) for 4 h at 37°C. After the incubation period, the plates were centrifuged (200 g) for 5 min to sediment the cells, the supernatant was removed and 25 µl of 0.2 N NaOH was added to each well. After 1 h, 200 µl of cold 15% trichloroacetic acid (TCA) was added to the wells and the samples were allowed to sit for 2–18 h at 4°C. The contents of the wells were then applied (under vacuum) to glass filters (Whatman GF/C). The filters were washed twice with 2 ml of 5% TCA and the radioactivity associated with the filters was measured by liquid scintillation spectroscopy.

Evaluation of apoptosis by flow cytometry
Cells subjected to different treatments as described were evaluated for apoptosis by multiparameter flow cytometric analysis using ethidium bromide (EtBr) uptake as previously described (21,22). Briefly, treated cells were washed by centrifugation, resuspended in PBS and stained with EtBr (4 µg/ml) on ice for 5–30 min. Cell death was evaluated by forward scatter (FSC) and EtBr uptake (detected by red fluorescence) using FACSort (Becton Dickinson Canada, Mississauga, Ontario, Canada).


    Results
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
The induction of apoptosis by CoA-IT inhibitors in arachidonate-depleted cell
In previous studies, we reported that the incubation of HL-60 cells with CoA-IT inhibitors blocks arachidonate–phospholipid remodeling and this cellular event is accompanied by an inhibition of cell proliferation and the onset of apoptosis (9,10). To begin to elucidate how the inhibition of arachidonate–phospholipid remodeling affects cell proliferation and apoptosis, HL-60 cells were cultured in serum-free, essential fatty acid-deficient media for at least 10 passages. This resulted in the generation of cells that contained far less arachidonate in glycerolipids than cells grown with serum as has previously been described (17). This provided a model to initially determine whether the presence of arachidonate in cellular lipids was crucial to the induction of apoptosis observed when cells are incubated with CoA-IT inhibitors. Figure 1Go shows that arachidonate-depleted cells were much less sensitive to the CoA-IT inhibitors SK&F45905 and SK&F98625 than were cells maintained in serum-free media supplemented with 3 µM AA. This observation suggested that there was something critical about the presence of arachidonate itself that was necessary for the induction of apoptosis.



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Fig. 1. Apoptosis of arachidonate-depleted and non-depleted HL-60 cells incubated with SK&F 45905 or SK&F 98625 for 24 h. HL-60 cells [that were maintained in serum-free media that was not supplemented (depleted) or supplemented with 3 µM arachidonic acid (non-depleted)] were incubated at 5x105 cells/ml for 24 h at 37°C in the presence of the indicated concentrations of SK&F 45905 or SK&F 98625. Cell death was assessed by forward scatter (FSC) and EtBr uptake (FL2). Values show the percentage of total cells in the two gated areas which represent live (R1) and apoptotic (R2) cells. The figure shows the results of one experiment which is representative of four separate experiments each performed in duplicate.

 
Distribution of [3H]arachidonate in HL-60 cells incubated with CoA-IT inhibitors
The metabolic events that occur as a result of blocking arachidonate–phospholipid remodeling in HL-60 cells labeled to isotopic equilibrium with [3H]AA were then examined. The cells were incubated with CoA-IT inhibitors for 24 h, then the distribution of [3H]arachidonate was measured in the different cellular and extracellular lipids. The most striking change that occurred as a result of blocking remodeling was an increase in the quantity of free [3H]AA found in the incubation medium of cells treated with the CoA-IT inhibitors. Figure 2Go shows that the treatment of HL-60 cells with two structurally distinct CoA-IT inhibitors results in the accumulation of free [3H]AA (not incorporated into glycerolipids) both inside and outside the cell after 24 h. The levels of free [3H]AA in the medium were 5–10-fold higher when cells were treated with CoA-IT inhibitors compared with non-treated cells. Although less striking, there was also an increase in intracellular free [3H]AA. Treatment of cells with CoA-IT inhibitors was previously shown to result in an accumulation of arachidonate associated with neutral lipids although the nature of these lipids was not determined (9). As shown in Figure 3Go, there was also an accumulation of [3H]arachidonate associated with both triacylglycerides and diacylglycerides in cells treated with CoA-IT inhibitors. These data suggested that as arachidonate remodeling between phospholipids is blocked (by CoA-IT inhibitors), cells lose their capacity to control free AA levels, and the resulting free arachidonic acid that is generated is either released from the cell as free AA or is acylated into TG. This shift of arachidonate into TG pools under conditions of high intracellular levels of AA has been observed in inflammatory cells and has been attributed to de novo glycerolipid biosynthesis (19,2325). Similar studies confirmed that this pathway is functional in HL-60 cells (data not shown).



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Fig. 2. The release of free [3H]arachidonic acid in cells incubated with CoA-IT inhibitors. HL-60 cells labelled to isotopic equilibrium with [3H]-arachidonate were incubated at 37°C for 24 h in the presence of 0.05% DMSO (v/v) (control), 50 µM SK&F 45905 or 25 µM SK&F 98625. The amount of radiolabel associated with cellular free fatty acids (inside cell) and free fatty acids in the incubation media (outside cell) were determined as described in Materials and methods. Values represent means ± SE for four separate experiments. aSignificantly different from control group as determined by analysis of variance using Fisher's least significant difference test, P < 0.05.

 


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Fig. 3. The distribution of [3H]arachidonate in lipid pools of HL-60 cells incubated with CoA-IT inhibitors. HL-60 cells labelled to isotopic equilibrium with [3H]arachidonate were incubated at 37°C for 24 h in the presence of 0.05% DMSO (v/v) (control), 50 µM SK&F 45905 or 25 µM SK&F 98625. At the indicated times, aliquots of cells were removed and the amount of radiolabel associated with cellular diacylglycerides (DG) or triacylglycerides (TG) was determined as described in Materials and methods. Values are expressed as percentages of total radioactivity in the incubations and represent means ± SE for four separate experiments. aSignificantly different from control group as determined by analysis of variance using Fisher's least significant difference test, P < 0.05.

 
Effect of CoA-IT inhibitors and exogenous fatty acids on cell proliferation and apoptosis
Since free AA was liberated from HL-60 cells when they were incubated with CoA-IT inhibitors, the next set of experiments were performed to determine whether the generation of free AA may be responsible for the anti-proliferative effect of CoA-IT inhibitors. As expected, the CoA-IT inhibitor SK&F 98625 dose-dependently inhibited cell proliferation as measured by the incorporation of [3H]thymidine into cellular DNA (Figure 4Go). Similarly, the incubation of HL-60 cells with arachidonic acid or with other long-chained polyunsaturated fatty acids like dihomogammalinolenic acid (20:3, n – 6) or docosatrienoic acid (20:3, n – 9) inhibited cell proliferation in a dose-dependent manner, although AA was more efficient than the trienoic acids. In contrast, an 18-carbon monounsaturated fatty acid, oleic acid, had no measurable effect on cell proliferation even when provided at concentrations of 100 µM.



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Fig. 4. The effect of the incubation of HL-60 cells with exogenous fatty acids and SK&F 98625 on [3H]thymidine incorporation. HL-60 cells (1x106 cells/ml) were incubated for 24 h with the indicated concentrations of arachidonic acid (AA), oleic acid (OA), dihomogammalinolenic acid (20:3, n – 6) (DGLA), eicosatrienoic acid (20:3, n – 9) (ETA) or SK&F 98625. The capacity of the cells to incorporate [3H]thymidine into cellular DNA as a measure of cell proliferation was then determined as described in Materials and methods. Values represent means ± SE of five to eight separate experiments each performed in triplicate.

 
The next series of experiments were designed to determine whether the anti-proliferative effect associated with elevated concentrations of AA was due to changes in levels of AA itself or metabolites generated from AA by the cyclooxygenase or 5-lipoxygenase pathways. Cells were incubated with the cyclooxygenase inhibitor, indomethacin, or the 5-lipoxygenase inhibitor, Zileuton, alone or in combination with exogenous AA or the CoA-IT inhibitor, SK&F 98625. Table IGo shows that these inhibitors of AA metabolism did not modify the antiproliferative effects associated with the incubation of HL-60 cells with either free AA or CoA-IT inhibitors. These data suggest that AA itself, and not oxygen-containing metabolites of AA, is responsible for the antiproliferative effect.


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Table I. The cyclooxygenase inhibitor indomethacin and the 5-lipoxygenase inhibitor Zileuton do not affect the proliferation of HL-60 cells
 
Arachidonic acid itself was also evaluated for its ability to induce apoptosis in HL-60 cells as assessed by flow cytometry; the incubation of HL-60 cells with 30–100 µM AA dose-dependently induced apoptosis in HL-60 cell as cells incubated with 30, 60 or 100 µM AA for 24 h showed 24 ± 8, 40 ± 10 and 70 ± 12% (means ± SEM, n = 4) of total cells as apoptotic after 24 h incubation, respectively.

The generation of ceramides in HL-60 cells incubated with AA or CoA-IT inhibitors
The release of AA following cPLA2 activation has recently been shown to be an important signaling mechanism for cell death in several cell lines and melanomas treated with TNF-{alpha}. AA accumulation in colon tumor cells treated with non-steroidal anti-inflammatory drugs has also been suggested to be responsible for the efficacy of these drugs in the inhibition of colorectal tumorigenesis (8). In both these forms of cell death the increase in free cellular AA has been shown to stimulate the conversion of sphingomyelin to ceramide, a known apoptotic signaling molecule. Therefore, as further evidence that CoA-IT inhibitors induce apoptosis via an increase in free AA, the ceramide content was determined in cells treated with CoA-IT inhibitors. Ceramides were measured by ESI-MS/MS which, unlike the commonly used diacylglycerol kinase method, allowed the measurement of individual molecular species of ceramides. As shown in Figure 5Go, the treatment of HL-60 cells with 60 µM AA or with 25 µM SK&F 98625 both resulted in an increase in cellular ceramide content. Importantly, the greatest increase in cellular ceramide content was in the 16:0 species of ceramides for both AA- and SK&F 98625-treated cells suggesting that both treatments caused the hydrolysis of the same pool of cellular sphingomyelin and, therefore, may both involve the same signaling mechanisms. In fact, this is the first report which identifies the individual molecular species of ceramides generated during the induction of apoptosis. One other report utilized similar methods, but failed to measure an accumulation of ceramides in Fas-treated T cell lines undergoing apoptosis (26)



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Fig. 5. The incubation of HL-60 cells with arachidonic acid or SK&F98625 induces the production of ceramides. HL-60 cells (0.5x106 cells/ml) were incubated for 18 h with 50 µM arachidonic acid or 25 µM SK&F98625. Cells were washed and counted, lipids were extracted and the different molecular species of ceramides were determined by ESI-MS/MS as described in the methods. Results are expressed as means ± SEM of four separate experiments each performed in triplicate. aSignificantly different from control group as determined by Fisher's least significant difference test, P < 0.05.

 

    Discussion
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
We have recently demonstrated that inhibiting the enzyme CoA-IT in HL-60 cells blocks the remodeling of arachidonate between phospholipid molecular species, resulting in a redistribution of cellular arachidonate pools (9). Interestingly, blocking this enzyme also attenuated cell proliferation and induced apoptosis (10). These findings raised the fundamental question of how blocking the remodeling of arachidonate affects cell proliferation and survival. Data in the current study reveal that the anti-proliferative effects are likely mediated by high levels of free AA that result from blocking the arachidonate remodeling pathway. This hypothesis is supported by the following five lines of evidence:
  1. The presence of arachidonate in cellular glycerolipids appears to be essential to maintaining the cell's sensitivity to the anti-proliferative effects of CoA-IT inhibitors.
  2. Blocking arachidonate–phospholipid remodeling leads to a marked accumulation of arachidonic acid on the outside of HL-60 cells and the accumulation of arachidonate-containing triglycerides within HL-60 cells. The accumulation of arachidonate-containing triglycerides appears to indicate that these cells have been exposed to high concentrations of AA.
  3. Twenty-carbon fatty acids such as arachidonic acid, eicosatrienoic acid and dihomogammalinolenic acid, but not 18-carbon fatty acids such as oleic acid attenuate cell proliferation in HL-60 cells.
  4. Inhibitors of AA metabolism via the cyclooxygenase and 5-lipoxygenase pathways do not influence the capacity of AA or CoA-IT inhibitors to attenuate cell proliferation.
  5. Both AA and CoA-IT inhibitors cause an increase in cellular ceramides which may be generated from the same cellular sphingomyelin pool as suggested by molecular species analyses using ESI-MS/MS.

Within the last few years, several studies have indicated that the control of arachidonic acid release may be important in signaling cell death. For example, cPLA2-mediated arachidonic acid release has been implicated in the signaling of TNF-{alpha} or Fas-mediated cytotoxicity in several fibroblast and melanoma cell lines, L929 fibrosarcoma cells and MCF7 human breast carcinoma cells (2,2734), as well as in the clonal deletion of thymocytes and immature B cells (35). In fact, both the present study and previous reports have shown that exogenous arachidonic acid itself can block cell proliferation and induce apoptosis (36). Other studies have put forward the thesis that cPLA2-mediated arachidonic acid release may be a necessary component in cell death signaling through the ceramide pathway (4,7,30). The link between the generation of free AA and the accumulation of ceramides in apoptotic cell death was recently extended in colorectal tumor cells where arachidonic acid accumulation in these cells following treatment with non-steroidal anti-inflammatory drugs was responsible for the generation of ceramides and ultimately apoptosis (8).

In the current study, AA accumulation following the inhibition of CoA-IT appears not to be due to an increase in cPLA2 activity (as suggested in other studies), but to a decrease in acylation activities that normally maintain low cellular levels of free AA (Figure 6Go). Normally, small amounts of free AA are constantly generated in the cell and this step is hypothesized to be mediated by the calcium-independent PLA2 (iPLA2) (37,38). This enzyme is thought to hydrolyse AA from 1-ether-linked phospholipids in resting cells (37,39). However, cellular AA is maintained at very low levels because it is then rapidly reacylated as arachidonoyl-CoA into 1-acyl-linked phospholipids. When cells are treated with CoA-IT inhibitors, there is an accumulation of arachidonate in 1-acyl-linked phospholipids and a depletion in 1-ether-linked phospholipids (9). This would be predicted since 1-acyl-linked and 1-ether-linked phospholipids are the respective donor and acceptor substrates in the CoA-IT-catalysed reaction (11,12). These observations indicated that, unlike more saturated fatty acid species which are not subjected to CoA-independent transacylation, the CoA-IT-driven remodeling pathway is the major pathway maintaining the equilibrium of arachidonate in cellular phospholipid species. Since this remodeling drives the formation of 1-acyl-2-lyso-phospholipids (via loss of arachidonate from the sn-2 position) and this substrate is necessary for the 1-acyl-2-lyso-phospholipid:arachidonoyl-CoA acyl transferase reaction, free AA released by iPLA2 activity cannot be reacylated if CoA-IT is blocked (Figure 6Go). Consequently, inhibiting the remodeling of arachidonate between phospholipids (utilizing CoA-IT inhibitors) leads to an accumulation of free AA by impeding the acylation/deacylation cycle driven by CoA-IT. This situation is exacerbated by the fact that neoplastic cells remodel arachidonate through phospholipids at rates many fold greater than their non-neoplastic counterparts: for example, the rate of arachidonate–phospholipid remodeling in P388D1 macrophages-like cells (40), HL-60 cells and monocyte-like THP-1 cells (41) is much greater than that observed in human neutrophils (11), cultured mast cells (42,43) or human lymphocytes (M.E.Surette, unpublished data). The enhancement of CoA-IT activity in non-neoplastic cells has been demonstrated, however, in TNF-{alpha}-treated human neutrophils indicating that this enzymatic activity is inducible (44).



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Fig. 6. Arachidonate–phospholipid remodeling and the consequences of blocking CoA-independent transacylase. The solid arrows show the arachidonate–phospholipid remodeling cycle driven by CoA-IT. The broken arrows show the consequences of blocking this cycle following the inhibition of CoA-IT: cells will accumulate free arachidonic acid which triggers the synthesis of arachidonate-containing TGs, the accumulation of cellular ceramides and the induction of apoptosis.

 
Although only small increases in free AA were measured within cells treated with CoA-IT inhibitors, two pieces of evidence indicate that important quantities of free AA were generated within these cells. First, large amounts (5–10 times more than control cells) of free AA were released by HL-60 cells into the incubation medium over a 24 h period when these cells were incubated with CoA-IT inhibitors. Secondly, there was an accumulation of arachidonate within cellular TG. Several studies have demonstrated (including this one) that when AA is presented to cells in large quantities, it is rapidly incorporated into DG and TG by de novo glycerolipid biosynthesis (19,2325). Therefore, even though the actual measured concentration of cellular free AA was only moderately increased in cells treated with CoA-IT inhibitors, the relatively large increases in arachidonate-containing triglycerides indicate that these HL-60 cells had been exposed to high intracellular concentrations of AA. These effects of blocking the remodeling pathway described herein are likely not limited to HL-60 cells since CoA-IT inhibitors also block arachidonate–phospholipid remodeling and induce apoptosis in human monocyte-like THP-1 cells, in MCF7 cells and in MDA231 epithelial breast cancer cells (41; F.H.Chilton, unpublished data).

A last set of experiments determined the potential for large quantities of AA to regulate the proliferation of HL-60 cells. Supplementation of the incubation medium with exogenous AA dose-dependently inhibited HL-60 cell proliferation and resulted in the induction of apoptosis. This effect was not seen after addition of oleic acid. The ability to tightly regulate cellular levels of free arachidonic acid therefore seems to be of importance in the control of cell proliferation. The production of oxygen-containing metabolites of the cyclooxygenase or lipoxygenase pathways are likely not responsible for the anti-proliferative effects of exogenous AA or CoA-IT inhibition since lipoxygenase or cyclooxygenase inhibitors did not affect cell proliferation. In fact, there are several recent reports in the literature which show that the production of oxygen-containing metabolites of AA may actually improve cell survival (5,6,45,46). For example, the overexpression of cyclooxygenase 2 in intestinal epithelial cells invokes a resistance to the induction of apoptosis that is reversed by cyclooxygenase inhibitors (6). Similarly, rat walker carcinoma cells whose major AA-derived metabolites are 12- and 15-HETE undergo time- and dose-dependent apoptosis when transfected with 12-lipoxygenase-specific antisense oligonucleotides, and this effect is mimicked by lipoxygenase inhibitors (5). While the release of free AA either following the blockage of the acylation/deacylation cycle with CoA-IT inhibitors or by activating PLA2 enzyme(s) appears to be important in blocking cell proliferation and inducing apoptosis, the subsequent metabolism of AA via lipoxygenases or cyclooxygenases appears to function as a cell survival mechanism. Whether AA metabolites are of themselves inducing a protective effect or are simply another mechanism by which cells may rid themselves of free AA remains to be determined. A recent report suggests that the induction of apoptosis in colorectal cancer cells by NSAIDs is, in fact, due to an accumulation of cellular AA and that the AA stimulates the conversion of sphingomyelin to the apoptosis-inducing ceramides (8). In the present study we have extended the association between the perturbation of the cellular control of AA levels, ceramide accumulation and apoptosis. In addition to accumulations of cellular free AA following the induction of cPLA2 by TNF-{alpha} or the inhibition of cyclooxygenases, we show here that, at least in HL-60 cells, the disregulation of AA remodeling by CoA-IT inhibitors also results in AA accumulation, ceramide generation and apoptosis. The ability to measure the individual molecular species of ceramides by ESI-MS/MS revealed that the ceramides generated directly by AA or following treatment with CoA-IT inhibitors may have been generated from the same sphingomyelin precursor pool, thereby supporting the link between CoA-IT inhibition, AA accumulation and apoptosis.

In conclusion, we have shown here that arachidonate–phospholipid remodeling is an important means of regulating free AA levels. The blockage of this pathway in HL-60 cells results in increased cellular free AA concentrations and concomitantly leads to an induction of apoptosis. These findings in conjunction with previous studies, which suggest that the susceptibility of cells to the induction of apoptosis is related to their capacity to mobilize free AA, further indicate that any perturbation in enzymatic mechanisms that regulate AA levels may affect cell proliferation and survival.


    Acknowledgments
 
The authors acknowledge the technical assistance of Dennis Swan (GC-MS) and Serge Picard (ESI-MS/MS). This work was supported in part by the National Institutes of Health grant AI24985 (to F.H.C.) and a grant from the Medical Research Council of Canada (to M.E.S.). M.E.S. is a Scholar of the Fonds de la Recherche en Santé du Québec.


    Notes
 
4 To whom correspondence should be addressed Email: marc.surette{at}crchul.ulaval.ca Back


    References
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 

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Received August 17, 1998; revised November 23, 1998; accepted December 2, 1998.