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Departments of Nutrition and * Biostatistics, School of Public Health and School of Medicine, University of North Carolina, Chapel Hill, NC 27599-7400
2To whom correspondence should be addressed. E-mail: steven_zeisel{at}unc.edu.
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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KEY WORDS: • apoptosis • choline • methionine • tryptophan • isoleucine
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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). Most apoptotic pathways involve either a receptor-linked or a DNA damage–induced (e.g., p53-inducible genes) death signal, an initiator cysteine protease (caspase), a mitochondrial-derived signal [e.g., cytochrome c release, reactive oxygen species (ROS)3
generation] under the control of regulatory factors (e.g., intracellular Ca ions, balance of antiapoptotic bcl-2 and proapoptotic bax proteins), and a final common apoptosis execution pathway involving activated caspases and DNA endonucleases (2
–4
). Apoptosis eliminates superfluous or damaged cells (5
) and is thought to be one of the fundamental mechanisms required for normal embryogenesis (6
–8
). Abnormally regulated apoptosis may result in fetal abnormalities, cancer and neurodegenerative diseases (9
). Therefore, factors that modulate apoptosis may have a role in both normal development and disease progression.
Although much underappreciated, alterations in nutrient availability can modulate apoptosis. For example, choline deficiency induces apoptosis in several cell types (10
–16
). Choline is a nutrient essential for methylation, phospholipid synthesis and neurotransmitter synthesis (17
). It seems likely that deficiency of other essential nutrients might also induce apoptosis. Methionine, like choline, is a donor of methyl groups, and their metabolic pathways are highly interrelated (18
). Tryptophan and isoleucine, like methionine, are essential amino acids required for protein synthesis, but they do not participate in methyl metabolism. In this study, we examined whether deficiency of a single essential amino acid (methionine, tryptophan or isoleucine) in cell culture induces apoptosis, and, if so, whether these nutrient deficiencies share common signaling components with choline deficiency–induced apoptosis.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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PC12 cells were the kind gift of J. P. O’Bryan at the University of North Carolina at Chapel Hill. Cells were maintained in Dulbecco’s modified Eagle’s Medium/Nutrient Mixture F-12 (DMEM/F12 medium; Atlanta Biologicals, Norcross, GA) containing 10% fetal bovine serum (Life Technologies, Grand Island, NY). Unless specified, PC12 cells were seeded at 106 cells/100-mm dish (Falcon, Franklin Lake, NJ) in medium containing serum for 48 h. The medium was replaced with a serum-free, chemically defined medium [DMEM/F12 with modified N2 medium supplement (14
)] for another 48 h. Cells were then incubated in the experimental media [the complete defined medium (control) or the same medium devoid of choline, methionine, tryptophan or isoleucine] for the indicated periods of time.
For primary neuronal cultures, fetal brains were derived from timed-pregnant Sprague-Dawley rats (Charles River, Raleigh, NC). Primary neurons, comprised mainly of postmitotic neurons from embryonic d-18 fetal rat cortex and hippocampus, were prepared and cultured as described previously (16
).
Determination of apoptosis.
For PC12 cells, apoptosis was assessed in both attached and detached cells, which were collected and deposited onto glass slides using a cytocentrifuge (Shandon, Runcorn, UK). Cells were fixed with methanol, stained with hematoxylin (Fisher, Fair Lawn, NJ), and mounted with Permount (Fisher). Slides were examined under a light microscope and the percentage of cells that were apoptotic was determined by counting at least 200 cells in four replicate cultures per treatment. Cells with fragmented nuclei (multiple, small hematoxylinophilic bodies) were defined as apoptotic. Primary cells grown on poly-D-lysine–coated glass chamber slides were fixed in 4% paraformaldehyde and then incubated with 1 µg/mL 4,6-diamidino-2-phenylindole (nuclear stain; Sigma Chemical, St. Louis, MO). The percentages of apoptotic cells were determined under a fluorescent microscope. Detached cells could be lost during this process. However, we found that the majority of cells remained attached after 48 h of nutrient deprivation. For DNA fragmentation (DNA ladders), samples harvested 72 h after treatment were assayed as previously described (14
).
Measurements of ceramide.
To determine the effect of nutrient deprivation on intracellular levels of ceramide, lipid was extracted and assayed using the HPLC method described by Previati, (19
) with some modifications (16
). Briefly, cellular lipids were extracted with methanol/chloroform (2:1, 1:1 and 1:2 sequentially). The extracts were combined, dried and derivatized with 10 µL of 100 mmol/L (S)-6 methoxy-
-methyl-2-naphthaleneacetic acid (Sigma), 10 µL of 100 mmol/L N, N' dicyclohexylcarbodiimide (Sigma) and 10 µL of 100 mmol/L 4-dimethylaminopyridine at -20°C for 3 h. The samples were then dried again and the derivatized products were extracted with 2 mL hexane and 5 mL methanol/water (4:1). After centrifugation (750 x g at 5 min), the upper phase was collected and the extraction procedure was repeated. The upper phases were then combined and dried. The dried samples were dissolved in 1 mL of hexane, and 50 µL of the dissolved samples was injected onto HPLC. Derivatized ceramides were resolved on an Econosphere CN, 250 x 4.6 mm column (Alltech, Deerfield, IL) equipped with a guard column with Discovery Cyano cartridge (Supelco, Bellefonte, PA). The column was initially equilibrated in 97% mobile phase A (hexane) and 3% mobile phase B (3% isopropanol in hexane). It was developed with a linear gradient to 10% B from 0 to 4 min, an isocratic run at 10% B from 5 to 10 min, and a linear gradient to 100% B from 11 to 18 min after sample injection. Ceramide concentration was calculated using a standard curve of known amounts of authentic ceramide.
Measurement of caspase activity.
Activation of certain caspases is considered a biochemical hallmark of apoptosis (20
,21
). These caspases may serve as initiators (e.g., caspase-8, -9, and -12) for apoptotic signaling as well as executors (e.g., caspase-3, -6, and -7) for the apoptotic machinery by cleaving intracellular substrates (22
). All caspases share a distinctive substrate specificity in that they recognize an aspartic acid residue at the cleavage site, whereas individual caspases may vary in their preference for amino acid residues neighboring the cleavage site. On the basis of their substrate specificity, a panel of peptide substrates and selective inhibitors for individual caspases was developed (23
). The activity of caspases was measured by a colorimetric protease assay (BioSource International, Camarillo, CA). In this assay, caspase activities that recognize the amino acid sequence, benzyloxycarbonyl aspartyl glutamylvalylaspartic acid (DEVD), benzyloxycarbonylvalylglutamylisoleucylaspartic acid (VEID), benzyloxycarbonylisoleucylglutamylthreonylaspartic acid (IETD) and benzyloxycarbonylleucylglutamylhistidylaspartic acid (LEHD) were measured using corresponding synthetic peptide substrates. These peptide substrates were labeled at their C-termini with para-nitroaniline (pNA). Cytosolic protein (from cells deprived of choline, methionine, tryptophan or isoleucine, or from cells of the time-matched controls) was prepared, and the caspase assay was performed based on the manufacturers instructions. Absorption of light by free pNA was quantified using a microtiter plate reader at 405 nm to assess the cleavage of the substrates by caspases.
Caspase inhibitor rescue experiments.
We determined whether caspase inhibitors (BioVision, Palo Alto, CA) prevented apoptosis induced by either choline or methionine deficiency. PC12 cells were grown in defined medium missing choline or methionine in the presence of the following: 1) choline or methionine [served as controls with complete nutrients and 0.5% dimethyl sulfoxide (DMSO), respectively]; 2) 0.5% DMSO (vehicle for delivering caspase inhibitors); 3) one of the following caspase inhibitors (100 µmol/L): broad-spectrum caspase inhibitors [Z-VAD- or Boc-D-fluoromethyl ketone (FMK)] or more selective inhibitors for single caspases (YVAD-, YDVAD-, DEVD-, WEHD-, VEID-, IETD-, LEHD-, or AEVD-FMK which inhibit caspase-1, -2, -3, -5, -6, -8, -9, and -10, respectively). Cells were harvested at either 60 h (choline deficient) or 36 h (methionine deficient) because choline deficiency requires more time than methionine deficiency to induce apoptosis. Experiments were performed in triplicate. Apoptosis was assessed as described earlier.
Measurement of protein synthesis.
The effect of nutrient deprivation on protein synthesis was assessed by measuring incorporation of 3H-leucine (Amersham Pharmacia Biotech, Buckinghamshire, UK) into proteins. Cells were treated as described earlier and exposed to 185 MBq/L 3H-leucine for 2 h before harvesting at 24, 48, and 72 h. An aliquot of sample was used for determining DNA content using a fluorometric method (24
); the remainder was dissolved in 0.1 mol/L NaOH. Macromolecules including protein were precipitated with addition of trichloroacetic acid (TCA, Sigma; final concentration 10%) and were collected with a glass fiber filter (Whatman, Kent, UK). The filter was washed with 10% TCA three times and scintillation fluid (Fisher) was added before the incorporated radioactivity was determined using a liquid scintillation counter (Wallac 1410, Pharmacia, Uppsala, Sweden).
Measurements of choline and its metabolites.
Samples were collected 48 h after cells had been treated with experimental media. DNA was measured as a basis for normalization (24
). After the addition of 14C-labeled internal standards, choline, phosphocholine and glycerophosphocholine in the aqueous phase of cell extracts (25
) were separated using HPLC (26
) and phosphatidylcholine in the organic phase was separated using TLC (26
). A [2H-methyl]-labeled internal standard for each metabolite was added to permit correction for recovery after analysis of choline moiety by a gas chromatography/mass spectrometry assay (26
).
Experiments on PC12 cells adapted to survive in low choline medium.
PC12 cells were gradually adapted to low choline (5 µmol/L choline) by stepwise withdrawal of choline from the medium (from 70 to 20 to 15 to 12 to 10 to 8 to 5 µmol/L choline) over a 2-mo period as described previously (27
). The selected cells (Weaned 5) were maintained in 5 µmol/L choline medium. They were split once each week in serum-containing medium (to facilitate cell attachment to matrix) and then changed back into low choline medium 3 h after seeding. For each experiment, they were grown with the same procedure used for the parental PC12 cells [in serum-containing medium for 2 d and then the complete (70 µmol/L choline) defined medium for another 2 d before changing into experimental medium].
To determine whether Weaned 5 cells were more resistant to choline deprivation–induced apoptosis than were the parental cells, both Weaned 5 cells and the parental cells were treated as described and then incubated with medium containing 0, 5 or 70 µmol/L choline for 72 h. Cells were harvested and apoptosis was determined as described above.
To determine whether Weaned 5 cells adapted to survive in low choline concentrations by correcting their intracellular concentrations of choline and its major metabolites, cells grown in control (70 µmol/L) or low choline (5 µmol/L) were collected for measurements. The parental cells were also included in this experiment for comparison.
To determine whether Weaned 5 cells also became more resistant to tryptophan deficiency, Weaned 5 and the parental cells were grown in an array of media in which tryptophan concentration had been serially diluted. Cells were harvested 72 h after the treatment and apoptosis was determined.
Statistics.
For experiments presented in Figures 1
, 2
, 4
, 8
, 9
and 10
, we used a two-way ANOVA with unbalanced data. The factors included main effects and interaction. Nonparametric one-way ANOVA was used for experiments in Figures 3
, 6
, and 7
due to concerns regarding distributional assumptions and homogeneity of variance between treatment groups.
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and 2
, treatment by time interactions were significant (P < 0.0001). For experiments presented in Figure 4
, the treatment by diet interaction was significant (P < 0.0003). For experiments presented in Figure 8
, the cell type by choline concentration interaction was significant (P < 0.0001). For experiments presented in Figure 10
, the cell by tryptophan interaction was significant (P = 0.0029). Thus, for experiments presented in Figures 1
and 2
, the treatment effect changed with time so that treatment effects were tested only at each time point (simple effects). For experiments presented in Figures 1
and 2
, comparisons between the control group and the rest of the treatment groups were performed at each time point using Dunnett’s critical difference test. For experiments presented in Figure 4
, treatment effects changed with diet so that treatment effects were tested only for each diet. For experiments presented in Figure 8
, cell type effects changed with choline concentration so that cell type effects were tested only for each choline concentration. For phosphocholine and glycerophosphocholine only experiments presented in Figure 9
, cell type effects changed with choline concentration so that cell type effects can be tested only for each choline concentration. For experiments presented in Figure 10
cell effects changed with the amount of tryptophan so that cell effects were tested only for each level of tryptophan. |
RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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More PC12 cells exhibited apoptotic morphology when cells were grown in a chemically defined medium devoid of choline, methionine, tryptophan or isoleucine than when grown in the same medium with complete nutrients (control, Fig. 1
). After 60 h of choline deprivation, the percentage of cells that were apoptotic was significantly greater than that in controls (9.8 ± 2.0% vs. 2.6 ± 0.6%, P < 0.05). Also, as early as 24 h after methionine, tryptophan or isoleucine deprivation, the apoptosis rate was significantly greater than that in controls (15.1 ± 2.8% vs. 0.43 ± 0.11%, P < 0.05, 44.4 ± 2.1% vs. 0.43 ± 0.11%, P < 0.01, or 10.8 ± 0.6% vs. 0.43 ± 0.11%, P < 0.05, different from time-matched controls, respectively). Apoptosis rates of nutrient-deprived cells increased over time throughout the experimental period, whereas that of control cells remained relatively constant (Fig. 1)
. The occurrence of apoptosis in the nutrient-deprived cells was further confirmed by characteristic DNA fragmentation (Fig. 1
, insert).
Deficiency of these single nutrients also induced apoptosis in primary neuronal cell cultures comprised mainly of postmitotic neurons from embryonic d-18 fetal rat cortex and hippocampus (Fig. 2
). Apoptosis rate was significantly greater than that in the time-matched control after 48 h of choline deprivation (12.8 ± 1.5% vs. 3.0 ± 0.7%, P < 0.05), 72 h of methionine deprivation (35.2 ± 6.3% vs. 6.8 ± 1.4%, P < 0.01) or 120 h of tryptophan deprivation (29.5 ± 3.2% vs. 11.1 ± 1.3%, P < 0.01).
Nutrient deficiencies increase ceramide levels.
We measured increases in ceramide concentrations when PC12 cells were deprived of any of the studied nutrients for 48 h (Fig. 3
). The ceramide level increased two- to threefold in cells deprived of methionine, tryptophan or isoleucine. Although the 30% increase in ceramide level after 48 h of choline deprivation was not significant (P = 0.058), a significant 52% increase in ceramide concetration was observed after 60 h of choline deprivation (76.2 ± 8.7 vs. 49.6 ± 3.1 pmol/µg DNA, P < 0.05). For each of the nutrient deficiencies, the extent of increase in ceramide concentrations correlated with the increase in apoptosis observed at 48 h.
Nutrient deficiencies activate caspases.
Caspase activities increased in choline-deficient PC12 cells as assessed by the cleavage of a panel of caspase substrates (Fig. 4
). When PC12 cells were deprived of choline for 60 h, all four substrates tested were cleaved, indicating the activation of more than one caspase. Caspases cleaving DEVD (mainly caspase-3 and -7) appeared to be most active. A similar profile of activation was found in a set of representative samples collected from PC12 cells deprived of a single essential amino acid (methionine, tryptophan or isoleucine) for 48 h (Fig. 4
, inset).
Broad-spectrum caspase inhibitors block nutrient deficiency–induced apoptosis.
To further support the observation that more than one caspase was involved, we determined whether suppressing caspases (using a series of synthetic peptides that inhibit broad-spectrum or specific individual caspases) inhibited choline deficiency–induced apoptosis in PC12 cells. Most cells grown in the complete medium had normal morphology (Fig. 5A
; control; 1.38 ± 0.21% cells were apoptotic). Cultures grown in choline-deficient medium had cells with typical apoptotic bodies (arrows in Fig. 5
B; 16.2 ± 1.1% cells were apoptotic, P < 0.01 different from control) at 60 h as expected. However, most cells grown in choline-deficient medium in the presence of Boc-D-FMK (a broad-spectrum caspase inhibitor) had normal morphology (Fig. 5
C; 3.6 ± 1.5% cells were apoptotic, not different from control) at 60 h. Similar results were observed in choline-deprived cells at 60 h treated with Z-VAD-FMK, another broad-spectrum caspase inhibitor (5.0 ± 0.7% cells were apoptotic, not different from control). DEVD-FMK, a reputed specific inhibitor for caspase-3 and -7, did not prevent choline deficiency–induced apoptosis (Fig. 5
D; 20.6 ± 3.8% cells were apoptotic, P < 0.01 different from control). Similar inability to prevent apoptosis was observed when other specific inhibitors for individual caspases (YVAD-, YDVAD-, WEHD-, VEID-, IETD-, LEHD-, and AEVD-FMK for caspase-1, -2, -5, -6, -8, -9 and -10, respectively) were used with choline-deprived cells (not shown). Experiments similar to those reported with choline-deprived cells also were reproduced with methionine-deprived cells. Most cells grown in complete medium had normal morphology (Fig. 5
E; control; 2.0 ± 0.2% cells were apoptotic), whereas more cells grown in methionine-deficient medium displayed apoptotic morphology at 36 h (Fig. 5
F; 15.3 ± 4.1% cells were apoptotic, P < 0.01 different from control). Most cells grown in methionine-deficient medium in the presence of Boc-D-FMK had normal morphology at 36 h (Fig. 5
G; 1.7 ± 0.3% cells were apoptotic, not different from control), whereas no inhibition of apoptosis was found when cells were grown in methionine-deficient medium in the presence of DEVD-FMK (Fig. 5
H; 22.3 ± 2.2% cells were apoptotic, P < 0.01 different from control) or IETD-FMK (not shown; 23.5 ± 2.5% cells were apoptotic, P < 0.01 different from control). Similarly, no inhibition of apoptosis was observed in cells grown in methionine-deficient medium in the presence of the other peptides that were specific inhibitors for individual caspases.
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In PC12 cells, after 48 or 72 h of choline deficiency, 3H-leucine incorporation, an indicator of protein synthesis, was not altered. In contrast, as early as 24 h after amino acid deficiency, 3H-leucine incorporation was decreased (Fig. 6
).
Amino acid deficiency does not alter choline metabolism.
Intracellular concentrations of choline and its major metabolites (phosphocholine, glycerophosphocholine and phosphatidylcholine) were diminished in choline-deficient cells, but most of these choline metabolites were unchanged in cells deficient in amino acids (Fig. 7
). Some modest decreases in phosphocholine concentrations were observed in methionine- and tryptophan-deprived cells (Fig. 7)
.
Cells that were resistant to choline deprivation were not resistant to tryptophan deprivation.
We reasoned that if nutrient deficiencies induce apoptosis through the same signaling pathway, resistance to one nutrient deficiency would confer resistance to other nutrient deficiencies. We selected a subclone of PC12 cells (Weaned 5) that survived in low choline medium (5 µmol/L choline), whereas the parental PC12 cells underwent apoptosis in this medium (Fig. 8
). Weaned 5 cells retained the capacity to undergo apoptosis when choline was completely removed from their medium (Fig. 8)
. The levels of choline and its metabolites decreased in Weaned 5 cells when grown in low choline medium in a manner similar to that observed in the parental PC12 cells (Fig. 9
). These data indicated that Weaned 5 cells did not adapt to low choline by correcting the choline metabolite abnormalities associated with choline deficiency. However, although these cells were resistant to choline deficiency–induced apoptosis, they were more sensitive than were the parental cells to tryptophan deficiency–induced apoptosis (Fig. 10
). Differences in proliferation rate of cells did not account for the difference in requirement for tryptophan; both Weaned 5 and PC12 cells had similar doubling times (
36 h) when they were grown in the complete defined medium.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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These observations are in keeping with Eagle’s work in the early 1950s showing the absolute requirements for these nutrients to support cell proliferation and prevent "degeneration" in culture (28
). We add to Eagle’s work by providing strong morphological and biochemical evidence demonstrating that specific nutrient deficiency caused cell death via a series of orderly events involving apoptosis rather than via a catastrophic disintegration (necrosis). Consistent with Eagle’s observations, the degree of sensitivity to deficiency of each tested nutrient appears to be cell type-specific (Figs. 1
, 2)
. PC12 cells were much more sensitive to tryptophan and methionine deficiencies than were the primary cells. After 24 h of tryptophan deprivation, 42.3% of PC12 cells were apoptotic compared with no significant increase in apoptosis in the primary cells. Similarly, 59.6% of PC12 cells were apoptotic after 72 h of methionine deprivation compared with 35.2% in the primary cells. On the other hand, PC12 cells were less sensitive to choline deficiency than were primary cells (Figs. 1
, 2)
. These differences in sensitivity could be the result of the different signaling pathways involved and the difference in intracellular milieus for signaling. However, they more likely reflect the difference between these two cell models in their relative requirement and initial intracellular pool of each tested nutrient. Although we showed that the effect of nutrient deficiency did not depend on cell cycle progression (because nutrient deficiencies induced apoptosis in postmitotic neurons), it is conceivable that requirements for different nutrients may vary when cells are at different stages of the cell cycle. PC12 cells may require more amino acids for protein synthesis as they progress through the cell cycle, whereas the differentiating neurons may need more choline for phosphatidylcholine synthesis as they undergo neurite outgrowth.
Ceramide accumulation and caspase activation were common features of choline and amino acid deficiency–induced apoptosis. These are well-described intermediate mediators of apoptosis signaling, and our observations suggest that these nutrient deficiencies share a common execution pathway. However, we also made two observations that are indicative of different signaling pathways invoked by the various nutrient deficiencies upstream of the ceramide/caspase components. First, choline deficiency and amino acid deficiencies did not share obvious metabolic consequences. Choline deficiency did not alter protein synthesis, and conversely, amino acid deficiency did not alter levels of choline and most of its metabolites. Second, Weaned 5 cells were resistant to choline deficiency–induced apoptosis but were still sensitive to tryptophan deficiency–induced apoptosis. To survive in low choline medium, Weaned 5 cells must have become resistant to apoptosis by modulating an apoptosis-mediating signal because they did not correct concentrations of choline and its metabolites. Whatever the mechanism involved, this adaptation did not render Weaned 5 cells resistant to tryptophan deficiency. This finding suggests the existence of different signaling triggered by tryptophan deficiency vs. choline deficiency.
Amino acid deficiency did not alter levels of choline and its metabolites except phosphocholine, whose levels were lower in tryptophan-deprived cells and, to a lesser extent, in methionine-deprived cells. Phosphocholine is produced mainly by phosphorylation of choline, a process that requires energy provided by ATP. It is possible that the decreased phosphocholine levels were due to energy depletion at the late stage of apoptosis found in these two conditions. However, this would not explain why phosphocholine levels were maintained in isoleucine-deprived cells when the percentage of apoptotic cells was also high. Phosphocholine is also derived from phospholipase C-mediated hydrolysis of phosphatidylcholine and sphingomyelin, a process that generates lipid-derived signaling molecules (diacylglycerol and ceramide, respectively). Perhaps the discrepancy in phosphocholine levels resulted from the different upstream signaling pathways initiated by each nutrient deprivation.
Caspases are activated in nutrient-deprived cells and caspase inhibitors can abort nutrient deficiency–induced cell death. These observations provide strong biochemical evidence to support the morphological finding that nutrient deficiency induces a specific intracellular program (apoptosis) rather than a generalized degeneration of the cell (necrosis) due to inability to maintain membrane integrity. Only broad-spectrum caspase inhibitors, but not more specific inhibitors of individual caspases, blocked nutrient deficiency–induced apoptosis. This finding indicates that multiple caspases are involved and that these caspases are capable of transmitting parallel signals. In addition, this finding also indicates the absence of a single "apex" caspase that is absolutely required for the initiation of the signal cascade. This is in contrast to the example typified by CD95-induced apoptosis in which blocking the initiator caspase, caspase-8, blocks the caspase cascade and apoptosis (29
,30
). However, we cannot exclude the possibility that specific inhibitors did not inhibit their target caspases completely and therefore failed to block apoptosis, although the dose used for several specific inhibitors was able to block the activities of their target caspases in vitro (31
,32
) and one of these specific inhibitors, DEVD-CHO (inhibitor specific for caspase-3–like caspases), prevents other stimuli (ceramide and thapsigargin)-induced apoptosis in the same cell line (33
,34
).
Our observations suggest that cells deprived of an essential nutrient die by apoptosis rather than by necrosis. Because the apoptosis mechanism involves removal of the cell before intracellular contents leak into the cell’s environment, death by this mechanism usually produces much less inflammation than does death by cell necrosis (35
). This may be why starved animals do not suffer from massive necrosis of tissues with subsequent harmful inflammatory responses, but rather gradually waste away, as organs lose cells, thereby sustaining remaining cells. Nutrient deprivation-induced apoptosis also has therapeutic implications; for example, L-asparaginase has been used to treat acute lymphoblastic leukemia (36
). We described previously how choline deficiency modulates apoptosis in the developing fetal hippocampus, resulting in permanent changes in brain function (11
,37
). Our current observations that amino acid deficiencies can also induce apoptosis suggest that these nutrients might influence brain development. Future studies may help us to better understand how the cell monitors nutrient status and how it triggers apoptosis when it first recognizes that it lacks a specific nutrient
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FOOTNOTES |
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3 Abbreviations used: DEVD, benzyloxycarbonyl aspartyl glutamylvalylaspartic acid; DMEM/F12, Dulbecco’s modified Eagle’s medium/nutrient mixture F-12; DMSO, dimethyl sulfoxide; FMK, fluoromethyl ketone; IETD, benzyloxycarbony lisoleucyl glutamylthreonylaspartic acid; LEHD, benzyloxycarbonyl leucyl glutamylhistidylaspartic acid; pNA, para-nitroaniline; ROS, reactive oxygen species; TCA, trichloroacetic acid; TD, tryptophan deprived; VEID, benzyloxycarbonyl valyl glutamylisoleucylaspartic acid.
Manuscript received 21 November 2001. Initial review completed 12 January 2002. Revision accepted 14 April 2002.
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LITERATURE CITED |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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