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(The FASEB Journal. 2000;14:2266-2276.)
© 2000 FASEB

H2O2-induced block of glycolysis as an active ADP-ribosylation reaction protecting cells from apoptosis

C. COLUSSI, M. C. ALBERTINI*, S. COPPOLA, S. ROVIDATI*, F. GALLI* and L. GHIBELLI1

Dipartimento di Biologia, Università di Roma Tor Vergata; 00133, Rome, Italy; and
* Istituto di Biochimica G. Fornaini, Università di Urbino, Rome, Italy

1Correspondence: Dipartimento di Biologia, Università di Roma Tor Vergata; via della Ricerca Scientifica, 00133, Roma. E-mail. ghibelli{at}uniroma2.it


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
H2O2 treatment on U937 cells leads to the block of glycolytic flux and the inactivation of glyceraldehyde-3-phosphate-dehydrogenase by a posttranslational modification (possibly ADP-ribosylation). Glycolysis spontaneously reactivates after 2 h of recovery from oxidative stress; thereafter cells begin to undergo apoptosis. The specific ADP-ribosylation inhibitor 3-aminobenzamide inhibits the stress-induced inactivation of glyceraldehyde-3-phosphate-dehydrogenase and the block of glycolysis; concomitantly, it anticipates and increases apoptosis. Exogenous block of glycolysis (i.e., by culture in glucose-free medium or with glucose analogs or after NAD depletion), turns the transient block into a stable one: this results in protection from apoptosis, even when downstream cell metabolism is kept active by the addition of pyruvate. All this evidence indicates that the stress-induced block of glycolysis is not the result of a passive oxidative damage, but rather an active cell reaction programmed via ADP-ribosylation for cell self-defense.—Colussi, C., Albertini, M. C., Coppola, S., Rovidati, S., Galli, F., Ghibelli, L. H2O2-induced block of glycolysis as an active ADP-ribosylation reaction protecting cells from apoptosis.


Key Words: NAD • oxidative stress • hydrogen peroxide • 3-aminobenzamide


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
THE MECHANISMS OF cell killing by oxidative stress, in particular by hydrogen peroxide, are not yet clarified, even though the effects of oxidative damage are well known: DNA breaks (1) , lipids peroxidation (2) , mitochondria failure (3) , alterations of calcium homeostasis (4) , actin reorganization (5 , 6) , nicotinamide adenine dinucleotide (NAD) depletion (7) , impairment of the energy metabolism (8) , glutathione depletion (9) , etc. Cells actively respond to oxidative stress by setting up many different reactions that increase cell defense or lead to adaptation to oxidant conditions (10) . It is often difficult to discriminate among the consequences of oxidative stress, between a direct radical damage and an active cell reaction to stress; as an example, stress-induced NAD depletion is not due to a direct radical effect on NAD, but is the result of the enzymatic NAD breakdown catalyzed by an hyperactivated poly(ADP-ribosyl)polymerase (7) . The strength of oxidative stress (i.e., the dose of H2O2) determines the type of cell death: the active process of apoptosis is triggered by lower doses, whereas the passive necrosis follows stronger ones (11) . Apoptosis is the cell response to sublethal damage, since the interference with the apoptotic process may lead to increase cell survival to stress even when the interfering agents do not reduce the extent of primary damage. Thus, in addition/alternative to the well-known self-protecting reactions (i.e., glutathione cycling in order to scavenge free radicals; ref 12 ), damaged cells are able to set up active self-destructive processes (apoptosis) in response to stress.

One of the consequences of oxidative stress is the block of glycolysis (8) ; this can be the consequence of the depletion of intracellular NAD pools [via the poly(ADP-ribosyl)polymerase (PARP) -catalyzed NAD breakdown] and/or of the inactivation of the glycolytic enzyme glyceraldehyde-3-phosphate dehydrogenase (GAPDH). This latter phenomenon is caused mainly by interferences of radicals with the cysteine residue present in the active site, which has been reported to be S-thiolated by hydrogen peroxide (13) and S-nitrosilated by nitric oxide (14) . Nitric oxide radicals can lead to GAPDH inactivation also by favoring the modification of the active site cystein residue by mono-ADP-ribosylation (15 , 16) .

ADP-ribosylations are reversible posttranslational modifications catalyzed by enzymes that add the ADP-ribose moiety of NAD onto acceptor proteins; this may result in the inactivation of the target protein (17) . PARP is a nuclear enzyme that can be activated by DNA breaks to build chains of NAD-derived poly(ADP-ribose) on acceptor chromatin proteins: one of its functions is the regulation of DNA repair (18 , 19) . In addition to PARP, eukaryotic cells possess also a number of mono(ADP-ribosyl)transferases in many cell compartments; many G-proteins are known substrates for mono(ADP-ribosylation) (20 21 22 23) , suggesting that intracellular signal transduction may be in part controlled by this type of modification. ADP-ribosylation events may be specifically inhibited by 3-aminobenzamide, effective on PARP at low doses, whereas higher concentrations are required to inhibit mono(ADP-ribosyl)transferases (24) . ADP-ribosylation reactions are involved in the cell response to oxidative stress: PARP is activated by radical-produced DNA breaks and may translate this type of damage into NAD depletion (25) and/or chromatin proteins modification. Moreover, it has been reported that proteins crucial for cell metabolism or structure such as GAPDH (26) or actin (27) may be mono ADP-ribosylated by oxidative stress.

We have been studying the mechanisms of oxidative stress-induced apoptosis, focusing on the role of PARP (11 , 28) and possibly mono(ADP-ribosylation) reactions (29) in the cells’ response to H2O2 treatments; in one of these studies, we reported that the ADP-ribosylation inhibitors 3-aminobenzamide (3ABA) and nicotinamide play double role in stress-induced apoptosis, being cell protective at the doses that inhibit only PARP but increasing apoptosis at the doses that inhibit also mono(ADP-ribosylations) (28) . In another study, we observed that the apoptotic process requires intracellular NAD in order to take place (29) . Stressed cells can actively (enzymatically) block their glycolytic flux with different modalities: inactivation of GAPDH and NAD depletion due to PARP activation (see above). This suggests that the two modalities, which occur through independent mechanisms, are in fact redundant ways to reach the same goal, implying that the stress-dependent block of glycolysis may be critical for cell repair/survival. This prompted us to investigate the stress-induced block of glycolysis, with the goal of understanding whether 1) it is an active cell reaction or a passive damage; 2) it is related to ADP-ribosylation, and in particular, mono(ADP-ribosylation); and 3) the block of glycolysis may have a role in stress-induced apoptosis.

In this study we show that glycolysis is transiently inhibited via ADP-ribosylation by oxidative stress in U937 cells through a 3-aminobenzamide sensitive block of GAPDH activity and that oxidative stress-induced apoptosis depends on an active glycolytic flux. This indicates that the endogenous block of glycolysis that follows oxidative stress may in fact be an active self-protective reaction of the cell rather than the passive result of an oxidative damage.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Cell culture
U937 cells were cultured in RPMI 1640 medium supplemented with 10% fetal calf serum (FCS), 2 mM L-glutamine, 100 IU/ml penicillin, and streptomycin) and kept in a controlled atmosphere (5% CO2) incubator at 37°C. Cell viability was assessed by trypan blue exclusion.

Oxidative stress
U937 cells were treated by adding to complete medium freshly prepared hydrogen peroxide at the concentration of 1 mM for 1 h; the cells were then washed and resuspended in fresh complete medium, and incubated for recovery. Apoptosis was measured at 5 h of recovery unless otherwise specified.

Treatments accompanying oxidative stress
ADP-ribosylation inhibition: 3-aminobenzamide (3ABA, 5 mM); inhibition of glycolysis: 2-deoxyglucose (DOG, 10 mM); pyruvate (10 mM); lactate (10 mM). 3ABA, lactate, and DOG were added 30 min before stress and added again in recovery (unless otherwise specified); pyruvate was added only in recovery.

Glycolysis inhibition in glucose-free medium: cells were placed in glucose-free RPMI 1640 supplemented with 10% FCS 1 h before oxidative stress. NAD depletion: U937 were cultured in 0.1 mM 6-amino nicotinamide for 3 days (>=90% NAD depletion) before performing an oxidative stress.

Induction of apoptosis by nonoxidative agents
Apoptosis was induced with the protein synthesis inhibitor puromycin (PMC, 10 µg/ml) or with the topoisomerase II inhibitor etoposide (VP16, 100 µg/ml). Both compounds were kept throughout the experiment. Apoptosis was measured at 4 h treatment; for glucose starvation, cells were placed in glucose-free medium 1 h before the apoptogenic treatment.

Analysis of apoptosis
Apoptosis was characterized by DNA fragmentation, which gives a ladder-like pattern, and nuclear fragmentation in several smaller fragments ranging in number from 2 up to >20 per cell, detectable by optical microscopy on slides of hematoxylin-stained cells or in Hoechst 33342 stained cell samples.

Preparation and staining of slides
2 x 105 cells, fixed in 4% paraformaldehyde, are loaded on a gelatinized slide, stained with hematoxylin, and analyzed for direct optical microscopy.

Analysis of DNA
106 cells are lysed as described (11) ; the purified DNA is loaded on a 1.5% agarose gel.

Quantification of apoptosis
The fraction of cells with fragmented nuclei among the total cell population is calculated on the hematoxylin-stained slides or in Hoechst 33342 stained cells, counting at least 200 cells in at least 6 random selected fields (11) . Apoptotic blebbing cells were detected at the contrast phase microscope, where they appear as raspberry-like cells; the fraction of blebbing cells is calculated by counting at least 200 cells in at least 6 random selected fields. Upon H2O2 treatment, apoptotic cells seldom develop blebs, whereas in the presence of 3ABA all apoptotic cells undergo strong blebbing (29) .

NAD measure
106 Trypan blue negative cells were lysed in Perchloric acid; supernatants of a 20' spin at 15 k rpm were analyzed in a coupled enzymatic reaction to measure NAD concentration spectrophotometrically as described in (30) . NAD concentration in control cells is 0.05 pmol/106 cells, ± 10%.

ATP measure
106 Trypan blue negative cells were processed according to the bioluminescent assay from Sigma (Technical Bulletin #BAAB-1); the extracts were analyzed in a luminometer.

Glycolytic flux measure
The glycolytic flux was estimated according to the rate of lactate extrusion and glucose consumption in a defined time interval by cells kept at the concentration of 1 x 106/ml. Briefly, 1 ml of medium containing 1 x 106 U937 cells was centrifuged to eliminate cells (5 min at 2000 rpm); lactate or glucose concentration in the supernatant was then measured. The first measure (zero time) was taken at the moment of medium change. For stressed cells, zero time coincided with the beginning of recovery.

Lactate was measured on samples utilizing a GM7 analyzer (Analox Instruments Ltd., London, U.K.). Briefly, samples were placed in the tubes supplied for Analox analyzer containing fluoride, heparin, and nitrite. Lactate content was determined by measuring O2 consumption by L-lactate:oxygen oxidoreductase. RPMI 1640 supplemented with 10% FCS that had not been in contact with cells gave values of 2.8 mM (blank), which was subtracted from the experimental values.

Glucose concentration was assayed measuring O2 consumption by glucose oxidase. RPMI 1640 supplemented with 10% FCS that had not been in contact with cells gave values of 10 mM ± 0.6 (10 mM is the nominal glucose concentration in RPMI 1640): this value has been considered as 100% in the experiments described.

GAPDH activity measure
Medium (1 ml) containing 106 cells was centrifuged to eliminate the medium; cells were then placed in 100 µl distilled water to produce a hypotonic lysis. The mixture was centrifuged and GAPDH activity in the cell extract was estimated by measuring NADH decrease in a coupled enzymatic reaction (NADH to NAD+ oxidation catalyzed by GAPDH) according to the spectrophotometric method described in A Manual of Biochemical Methods (by F. Beutler, Grunc and Strutton, New York), measured in the supernatant by a spectrophotometric assay involving two coupled reactions. The optical density decrease of the system was measured against that of the blank at 340 nm at 37°C for 10 to 20 min.

Phosphodiesterase assay
Incubation of cell extracts with phosphodiesterase may reverse GAPDH inactivation due to ADP-ribosylation but not that due to mere cystein oxidation (31) . Thus, the type of inhibition of H2O2-induced GAPDH activity was investigated by the following phosphodiesterase assay: 106 cells were washed and resuspended in phosphodiesterase-permeating buffer (0,1M HEPES NaOH; 1 mM MgCl2, pH=7.8) plus or minus 50 µg/ml phosphodiesterase, during 1.5 h at 4°C, according to ref 32 . The cells were then washed in phosphate-buffered saline and lysed with 100 µl of distilled water. The lysate was used to measure GAPDH activity as described.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Hydrogen peroxide treatment blocks glycolysis
U937 cells are human promonocytes derived from a histiocytic lymphoma. Their origin renders them interesting for the study of hydrogen peroxide induced apoptosis. Our protocol for the oxidative treatment was a stress period (1 h), after which cells are washed, resuspended in fresh medium, and followed for recovery. The reason for choosing this protocol with respect to a continuous stress treatment is that the continuous stress does not allow apoptosis to take place, leading instead to cell death by necrosis (not shown).

Here we compared the rate of glycolytic flux in stressed and untreated cells. Exponentially growing healthy U937 consume 39 µmol of glucose and produce 34 µmol of lactate per hour per 108 cells (Table 1 ). U937 cells were then treated with 1 mM hydrogen peroxide for 1 h, then cells were washed and resuspended in fresh complete medium for recovery. In the first hour of recovery, stressed U937 cells failed to accumulate lactate or consume glucose, showing that the glycolytic flux is in fact blocked (Table 1) .


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  		Table 1. Effect of H2O2 on glycolytic flux, intracellular NAD, and GAPDH activity in U937 cellsa

GAPDH is inactivated by hydrogen peroxide
We investigated two possible mechanisms for H2O2-induced glycolytic block. First, NAD depletion due to PARP activation by the DNA breaks created by oxidative stress; however, NAD levels were not affected by oxidative stress (Table 1) . Second, we analyzed the activity of GAPDH after H2O2 treatment: we found that GAPDH is inactivated by the hydrogen peroxide in U937 cells (Table 1) .

To check whether the inactivation of GAPDH is the inevitable consequence of any block of glycolysis, we interfered with the glycolytic flux with the glucose analog 2-deoxyglucose (10 mM) or with the drug cytochalasin B (5 µg/ml), which is known to inhibit glucose transport across plasma membrane (33) ; these treatments reduced glycolysis by 71% and 85%, respectively, but GAPDH was not inactivated (Fig. 1 ); thus, GAPDH inactivation is not the mere consequence of a block of glycolysis.



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  		Figure 1. GAPDH is a target of H2O2 inactivation only when the glycolytic flux is active. The effect of the glucose analog DOG and CCB on lactate production and GAPDH activity were measured in healthy (untr.) and stressed cells. Healthy cells were placed in fresh medium for 1 h prior to measurement; for stressed cells; measurements were taken at 1 h of recovery from H2O2 treatment (see Materials and Methods). DOG and CCB strongly reduce lactate production without inactivating GAPDH in healthy cells. In stressed cells, DOG and CCB do not allow H2O2-dependent GAPDH inactivation. The values reported are calculated as percent of control values (for absolute values of control, see Table 1 ) and are the mean of three independent experiments ± SD. untr., healthy cells; CCB, 5 µg/ml cytochalasin B; DOG, 10 mg/ml 2-deoxyglucose

We next investigated whether GAPDH needs to be involved in glycolysis in order to be inactivated by H2O2. We found that in the presence of 2-deoxyglucose or cytochalasin B, hydrogen peroxide was no longer able to inactivate GAPDH, whose activity resulted instead slightly increased (Fig. 1) . This result shows that the enzyme must be actively involved in the glycolytic flux in order to be target of oxidative inactivation.

GAPDH inactivation in stressed cells is due to a precise posttranslational modification, possibly being an ADP-ribosylation
It is known that GAPDH can be inactivated by oxidative treatments—direct oxidation or nitrosilation of the cystein in the active site (13 , 14) ; alternatively, the cystein residue can be modified by ADP-ribosylation, thereby inactivating the enzyme (15) . This latter phenomenon can be reversed, by in vitro reaction, by phosphodiesterase, an enzyme that is able to promote the detachment of the cystein-bound, covalently linked ADP-ribose from the acceptor proteins (32) , thus restoring enzyme activity (31) . To discriminate between the different mechanisms of inactivation, we treated extracts of control and H2O2-treated cells with phosphodiesterase. Indeed, if GADPH inactivation is mediated by ADP-ribosylation, this procedure would lead to in vitro reactivation of the enzyme, whereas a direct oxidation of the cystein residues will not be reversed. Table 2 shows that GAPDH activity in extracts from stressed cells nearly recovers control values upon incubation with phosphodiesterase, whereas the enzyme had no effect on the activity of GAPDH from control cells. The ‘reactivability’ of GAPDH shows that the enzyme is not damaged by oxidative stress, but rather is subjected to a precise posttranslational modification, very likely an ADP-ribosylation.


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  		Table 2. GAPDH reactivation by phosphodiesterasea

The presence of 3-aminobenzamide rescues GAPDH activity and glycolysis in stressed cells
Next, we analyzed whether 3ABA, a specific inhibitor of ADP-ribosylation processes, was able to avoid H2O2-dependent GAPDH inactivation. Figure 2 shows that 3ABA indeed reduces the oxidative-induced GAPDH inactivation and concomitantly allows glycolysis to take place in stressed cells (albeit at a lower rate), thus reinforcing the link between the stress-induced block of glycolysis and GAPDH inactivation. 3ABA has no effect on glycolysis or GAPDH activity in unstressed cells, indicating that the compound by itself has no direct effects on the glycolytic flux. These results show that 3ABA inhibits H2O2-dependent GAPDH ADP-ribosylation, possibly via a direct effect.



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  		Figure 2. Effect of 3-aminobenzamide on H2O2-induced glycolytic alterations. Glucose consumption, lactate production, and GAPDH activity in healthy (untr.) and stressed U937 cells, ± 5 mM 3-aminobenzamide (3ABA). Healthy cells were placed in fresh medium for 1 h prior to measurement; for stressed cells, measurements were taken at 1 h of recovery from H2O2 treatment (see Materials and Methods). 3ABA reduces H2O2-dependent GAPDH inactivation and concomitantly allows glycolysis to take place. However, it does not increase the glycolytic flux on healthy cells. The values are the mean of three independent experiments ± SD.

The oxidative block of the glycolytic flux is transient; stress-induced apoptosis begins after the block is reversed and is increased by 3-aminobenzamide
The stress-induced block of glycolysis is spontaneously reversed after 2 h of recovery from the oxidative treatment (Fig. 3A ); from that time on, the extent of the flux is similar to untreated cells.



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  		Figure 3. Temporal relation between glycolysis and H2O2-induced apoptosis. A) Lactate accumulation in healthy (untreated) and stressed cells. Time zero indicates medium change, which coincides with the recovery period for stressed cells. The values indicate µmol/108 cells. The stress-induced block of glycolysis is spontaneously reversed after the first 2 h of recovery from the oxidative treatment. A similar kinetics was observed in two additional experiments. B) Compared kinetics of apoptosis and lactate accumulation after H2O2 treatment. Apoptosis begins when glycolytic flux is restored (squares, apoptosis; circles, lactate). C) Effect of 3-aminobenzamide (3ABA) on H2O2-induced apoptosis: when glycolysis is not blocked (see Fig. 2 ), apoptosis is increased and anticipated. One of >10 experiments is shown.

Figure 3B shows the kinetics of apoptosis induced by H2O2 compared to the kinetics of glycolysis. Curiously, stress-induced apoptosis begins just after the restoration of a normal glycolytic flux; in cells stressed in the presence of 3ABA, where glycolysis is not blocked, apoptosis is increased and anticipated (Fig. 3C and ref 29 ): these observations suggest a possible correlation between apoptosis and presence of an active glycolytic flux and prompted us to analyze in detail the possible dependance of apoptosis on glycolysis.

Treatments that reduce glycolysis reduce the extent of stress-induced apoptosis
The observation that apoptosis begins only when the stress-induced block of glycolysis is reversed suggested we check whether an exogenously induced block, by turning stable the transient block of glycolysis might protect stressed cells from apoptosis.

Glycolysis was blocked by the addition of the glucose analog 2-deoxyglucose (10 mM) or by culture in glucose-free medium; these treatments reduced the glycolytic flux in healthy cells by 50 to 80%; ATP levels were lowered by only <= 20% with respect to control levels after 24 h treatment and no cytotoxicity was detectable after up to 48 h of glucose starvation or 2-deoxyglucose treatment (not shown). Glycolysis was also blocked in healthy cells by 100% by depleting the intracellular NAD pool with the nicotinamide analog 6-amino nicotinamide, which reduced NAD levels of >= 90% with respect to control in 3 days; these cells were perfectly viable even though they stopped growing within 24–48 h.

Figure 4A shows that upon oxidative stress, the treatments that exogenously block glycolysis do not allow the restoration of the glycolytic flux after 2 h of recovery.



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  		Figure 4. The exogenous block of glycolysis reduces H2O2-induced apoptosis. U937 cells were treated with H2O2 in the presence of 10 mM 2-deoxyglucose (DOG), cultured in glucose-free RPMI medium (-glu), or depleted of NAD by 0.1 mM 6-amino nicotinamide (+6AN), as described in Materials and Methods. A) Time course of lactate accumulation during recovery from H2O2 in cells stressed with or without the above-mentioned glycolytic blocking treatments. Lactate production is restored in stressed cells (see also Fig. 3 A), whereas in the presence of the inhibitors it remains blocked throughout the experiment. B) The extent of apoptosis measured at 5 h of recovery from H2O2 treatment in the presence or absence of the glycolytic inhibiting treatments. In all cases, the permanence of the glycolytic block is associated with a reduction of apoptosis.

In all these instances, cells with a reduced glycolytic flux were protected from stress-induced apoptosis, as shown in Fig. 4B . This was not a shift of the type of cell death from apoptosis to necrosis, since it was not accompanied by any increase in trypan blue or propidium iodide permeability up to 24 h of recovery (not shown).

These results suggests that the glycolysis inhibitors exert their anti-apoptotic effect by turning a transient glycolytic block into a permanent one. To be protective, the glycolysis inhibitors should be required only when the glycolytic flux is going to be restored, that is, at >=2 h of recovery from stress. To demonstrate this, we set up several experimental schemes where glycolysis is exogenously blocked by DOG at different periods of the oxidative treatment: pretreatment, stress, recovery. Apoptosis was then evaluated at 5 h of recovery (Fig. 5 ). The result of this experiment indicates that DOG must be present during recovery in order to exert its protective action, whereas its presence as a pretreatment or during the stress does not affect H2O2-induced apoptosis.



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  		Figure 5. The exogenous block of glycolysis exerts a protection from apoptosis during recovery. The figure describes several experimental schemes, where glycolysis is exogenously blocked by 2-deoxyglucose (DOG) at different periods of the oxidative treatment, i.e., pretreatment, stress, and recovery. The times of DOG addition (+) are shown; (-) indicates the absence of DOG. The percentage of apoptosis for each different type of treatment (average of 3 to 5 experiments ±SD) is calculated at 5 h of recovery from H2O2. The experiments show that the protective effect of DOG does not depend on pretreatment or on its presence during the stress, but instead coincides with the presence of DOG during recovery, when glycolysis is usually resumed.

3ABA exerts its proapoptotic activity only in cells with an ongoing glycolytic flux
The results just described point to a role of an ongoing glycolytic flux in order to develop H2O2-induced apoptosis. This would imply that by inhibiting GAPDH inactivation, 3ABA increases apoptosis because it does not allow the H2O2-dependent block of glycolysis. If this is true, then it would follow that 3ABA should be no longer able to affect H2O2-induced apoptosis in stressed cells where glycolysis is blocked by other means, i.e., DOG addition. This is indeed what occurs. Table 3 shows that 3ABA fails to exert a proapoptotic activity in cells stressed in the presence of DOG. It also fails to induce the peculiar blebbing morphology that we have shown to be strictly associated with the extra apoptosis induced by 3ABA (29) . This indicates that the proapoptotic effect of 3ABA is exerted only in cells with an ongoing glycolytic flux, showing that the target of the proapoptotic action of 3ABA is glycolysis.


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  		Table 3. 3ABA exerts its proapoptotic activity only in cells with an ongoing glycolytic fluxa

Protection from apoptosis is also achieved when the block of glycolysis does not affect downstream energy metabolism
The data shown above indicate that a block of glycolysis protects cells from H2O2-induced apoptosis. To understand whether the protective effect is due to the block of glycolysis in itself or to a consequent reduction of the downstream energetic metabolism, we designed two experimental approaches that allowed stressed cells to maintain active the downstream energy metabolism on oxidative stress when glycolysis is blocked. In the first approach, glycolysis was blocked by glucose-free medium, and 1 mM pyruvate was added during recovery from stress (pyruvate was added only during recovery, because of its ability to scavenge H2O2 as an {alpha}-ketoacid; refs 34 , 35 ) to feed the downstream metabolism. As shown in Fig. 6 , 1 mM pyruvate does not revert the protective effect of glucose starvation, indicating that the protection is not due to the block of downstream metabolism. In the second approach, glycolysis was blocked by retroinhibition with the end products lactate (10 mM) or pyruvate (10 mM), thus allowing the downstream metabolism unaffected. In this experiment, we found that the ongoing downstream metabolism did not affect the protective effect of glycolytic block.



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  		Figure 6. The protective effect of the block of glycolysis is not dependent on a secondary effect of downstream energy metabolism. The presence of lactate or pyruvate, which block glycolysis by retroinhibition without affecting downstream energy metabolism, does not reduce the protective effect of the glycolytic block. The same result is obtained when 1 mM pyruvate is added in the glucose-free medium. Apoptosis was measured at 5 h of recovery; the reported values are the average of three independent experiments ± SD. lact = 10 mM lactate; pyr = 10 mM pyruvate; -glu = glucose-free medium.

These experiments show that the cell protective effect is due to the block of glycolysis in itself.

The block of glycolysis protects U937 cells also from apoptosis induced by nonoxidative agents
The process of apoptosis may be logically subdivided into two subsequent phases: induction and signaling. The different reactions triggered by various inducers (induction phase) converge into the mainstream of the signaling cascade (signaling phase), which is the intrinsic mechanism of apoptosis, occurring independently of the inducer used (36 , 37) .

To understand whether the block of glycolysis interferes with the mechanism of apoptosis induction by H2O2 or with the intrinsic process of apoptotic signaling, we performed experiments on U937 where apoptosis was induced by agents acting in different ways, such as the protein synthesis inhibitor PMC (10 µg/ml) or the topoisomerase II inhibitor etoposide (VP16, 100 µg/ml). These inducers share with H2O2 the steps of the intrinsic apoptotic signaling, whereas the upstream induction phases are carried out differently. We blocked glycolysis either by adding DOG to the regular medium or by culturing cells in glucose-free medium. As shown in Fig. 7A , the block of glycolysis led to a significant reduction of apoptosis induced by both apoptogenic agents, thus indicating that the block of glycolysis interferes with the apoptotic signaling, i.e., with the intrinsic mechanism of apoptosis.



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  		Figure 7. Effect of glycolysis block on apoptosis induced by nonoxidative agents. Apoptosis was induced on U937 cells with puromycin (PMC) or etoposide (VP16) as described in Materials and Methods. Cells were transferred in glucose-free medium 1 h before the apoptogenic treatment. Apoptosis was measured at 4 h of treatment. Block of glycolysis by treatment with 10 mM DOG exerted a similar protective effect (not shown); the reported values are the average of three experiments ± SD.


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
In this study, we propose that the oxidative stress-induced block of glycolysis is not a passive oxidative damage but an active cell reaction set up for cell protection. Indeed, the elimination of the glycolytic block (with the ADP-ribosylation inhibitor 3ABA) increases apoptosis, whereas the prolongation of the block (with glucose starvation) instead has an anti-apoptotic effect. One could speculate that 3ABA might increase stress-induced apoptosis through other means, i.e., unrelated to glycolysis. However, this is very unlikely. Indeed, the block of glycolysis occurs via ADP-ribosylation of GAPDH (Table 2) ; moreover, in DOG-treated cells, where glycolysis is inhibited at a step upstream to GAPDH, 3ABA does not exert any proapoptotic effect (Table 3) , showing that the target of the proapoptotic action of 3ABA is glycolysis.

The main message from this study is that the block of glycolysis on hydrogen peroxide treatment is not a passive oxidative damage but an active cell reaction made for self-protection, as though cells put themselves in a state of stand-by waiting to decide whether repair the damage and survive or commit suicide by apoptosis. To make a bit of teleology, the decision is important, and the time cells take for it may turn out to be well spent: on one side, cell loss is always an energetic burden for the organism; on the other side, cells with damaged DNA are dangerous because they are potentially mutated and transformed, and their elimination may be a good choice in the long run. The block of glycolysis can be actively (enzymatically) reached in different cell systems with different modalities: inactivation of GAPDH and NAD depletion due to PARP activation. This suggests that the two modalities, which occur through independent mechanisms, are in fact redundant ways to reach the same goal, implying that the stress-dependent block of glycolysis may have a crucial importance for cell repair/survival. In cells where H2O2 activates PARP-mediated NAD depletion, NAD levels are restored during recovery; only then does apoptosis begin to take place (28) . This is reminiscent of the occurrence of apoptosis only when glycolysis is restored (this study, Fig. 3B ). It has been observed that NAD depletion is incompatible with apoptosis, leading instead to necrosis (28) or to increased survival to cell damage (38) . We may speculate that the mechanism through which NAD is required for apoptosis is by allowing the glycolytic flux.

3ABA is usually considered quite a specific inhibitor of the ADP-ribosylation processes (24) . It has often been used as a tool to analyze PARP involvement in apoptosis (11) ; however, the doses of 3ABA commonly used for these experiments are high enough (>=2–3 mM) to also inhibit mono(ADP-ribosylations) (24) . A previous study of ours (29) showed that whereas low (<1 mM) 3ABA doses have a cell protective role in H2O2-induced apoptosis, higher doses exert instead a proapoptotic effect on stressed cells, implying that mono(ADP-ribosylations) may play a role in the cell antioxidant defense system. We show here that in intact cells, GAPDH is inactivated by H2O2 via a posttranslational covalent modification (probably an ADP-ribosylation) and that in the presence of 3ABA the inactivation hardly occurs. It is thus possible that 3ABA directly inhibits GAPDH modification; however, we cannot rule out that 3ABA may inhibit another hypothetical ADP-ribosylation event, upstream with respect to GAPDH modification, which is the direct responsible for the effect on GAPDH. Mono-ADP-ribosylation of GAPDH is reported in the literature, such as that induced by the Golgi transport inhibitor brefeldin A (39) ; this occurs through an enzymatic ‘canonic’ modification event, which might possibly be 3ABA sensitive. GAPDH was also found ADP-ribosylated during the physiological process of hibernation (31) and after incubation with nitric oxide, which modifies a cysteine present in the active site (15) ; this is not a ‘canonical’ mono-ADP-ribosylation, being instead an automodification favored by a previous S-nitrosylation (40) (it is not clear even if it is really an ADP-ribosylation or rather a covalent attachment of the whole NAD+ or NADH molecule; ref 41 ). The ADP-ribose can be detached from the modified proteins by incubation with phosphodiesterase, through enzymatic release of AMP (32) ; the ribose moiety is then liberated, possibly through an autocatalyzed process, to restore a functional active site (31) . It will be interesting to investigate whether H2O2-induced GAPDH modification may be sensitive to 3ABA inhibition; indeed, so far the mono-ADP-ribosylations reported to be sensitive to 3ABA are modifications of arginine rather than cystein residues (24) . Studies are progressing in our laboratory to understand whether nitric oxide may mediate H2O2-induced GAPDH ADP-ribosylation and to clarify the mechanism of 3ABA inhibition of GAPDH inactivation.

It has long been known that tumor cells have an altered glycolytic metabolism, with an increased flux rate (42) ; they may survive to low oxygen tension (i.e., in the absence of oxygen-carrying blood vessels) with the so-called ‘anaerobic glycolysis’, process that turns pyruvate into lactate. In some tumor cells the citric acid cycle is fed by lipids and amino acids rather than by the glycolysis-produced pyruvate, and the pyruvate dehydrogenase complex, which links glycolysis with the downstream energy metabolism, is sometimes missing (43) , thus dissociating the main pathways of energy production. It follows that in different tumor cell lines the differences in glycolytic metabolism may lead to different consequences of glucose starvation. In the U937 cells examined in this study, glucose deprivation by itself is nonlethal in the first 48 h of starvation, after which cells begin to gradually undergo apoptosis; concomitantly, no drop of ATP occurs, and after 24 h ATP levels are almost 80% with respect to untreated cells. Instead, in the human leukemic cells CEM, glucose starvation leads to a sudden ATP depletion and to cell death by necrosis within 24 h (44) . Thus, the energetic behavior of U937 seems ideal to study the consequences of glycolysis block on cell metabolism without affecting the cells’ energy supply.

It emerges from this study that an ongoing glycolytic flux favors H2O2-induced apoptosis. Several obvious possible explanations for this phenomenon were analyzed, but could be excluded on the basis of experimental observations. First, glycolysis contributes to the cells’ energy level and apoptosis is (generally) an energy-requiring process, since damaged, de-energized cells die instead by necrosis; however, in our system a glycolytic block and/or glucose starvation do not significantly alter ATP levels. Second, glycolysis produces pyruvate, which is the fuel for the mitochondrial energy metabolism (citric acid cycle and oxidative phosphorylation), and it is well known that active mitochondria are crucially involved in apoptotic cell signaling (45) ; however, the maintenance of the downstream metabolism by the addition of pyruvate did not alter the anti-apoptotic effect of the glycolytic block.

We have recently shown that apoptosis occurs via redox modulation even when the inducers are not redox related: this is achieved by the active extrusion of reduced glutathione, which leads to a shift in the cell redox status (46 47 48) . We provide evidence (Fig. 7) that the anti-apoptotic effect of the block of glycolysis is not restricted to oxidative stress-induced apoptosis, implying that it affects the apoptotic process in itself rather than the signaling pathway triggered by radical damage. Preliminary data in our laboratory suggest a possible connection of glycolysis and glutathione depletion in apoptosis (not shown). Speculation about these mechanisms can be subdivided into two logic subgroups. On the one hand, it is possible that an ongoing glycolytic flux may actively favor the onset of apoptosis: one of the intermediate glycolytic products may facilitate some of the steps of the apoptotic intracellular signaling. On the other hand, one might conceive that a block of glycolysis might free the glycolytic enzymes from their normal tasks, allowing them to perform alternative, anti-apoptotic cell-protective functions. Indeed, some of the glycolytic enzymes are known to play double roles in the cell. As an example, it has been reported that GAPDH has many functions unrelated to glycolysis. Among the three GAPDH isoform in Saccharomyces cerevisiae one is a stress-responsive protein required for survival to heat shock, and is never involved in glycolysis (49) . GAPDH has been identified as a transporter of nucleotides in synaptic vesicles (50) ; it may bind specific tRNAs (51) and DNA (52) , where it is able to activate transcription (53) ; it is also reported to be involved in nucleotides metabolism (54) . Particularly important for the phenomena described in the present work may be its interactions with cytoskeleton; indeed, GAPDH may interact with band 3, an abundant anion exchanger protein present in the erythrocytes’ cytoskeleton (55) , and with microtubules (56) . This ability is shared by phosphofructokinase, which may also bind to microtubules (57) . These alternative, nonglycolytic roles may have the function of contrasting the apoptotic process. It is conceivable that the binding of glycolytic enzymes to cytoskeleton may help to protect cellular structures. In the case of apoptosis, binding to the tubulin or actin cytoskeleton may protect the cell body from apoptotic blebbing, which in some instances may be a causative event of apoptosis (29) .

In conclusion, we want to stress that it is often difficult to discriminate among the consequences of oxidative stress, between a direct radical damage and an active cell reaction to stress. In this view, the block of glycolysis is often considered as mere oxidative damage. We hope that our results showing that the block of glycolysis is instead a mechanism of cell-protection from oxidative stress will help provide further impetus to this field, helping to elucidate our still incomplete understanding of the strategies of cell repair/survival/apoptosis.


   ACKNOWLEDGMENTS
 
We wish to thank Prof. F. Canestrari and Prof. A. Accorsi for helpful discussions. The work was partly supported by a grant from the Italian National Research Counsel (Contributo CNR99.02491.CT04).

Received for publication March 6, 2000. Revision received April 24, 2000.
   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
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