Journal of Neurochemistry, 2001, 78, 265±275
Oxidative stress mediates neuronal DNA damage and apoptosis in response to cytosine arabinoside Herbert M. Geller,* Ke-Yi Cheng,* Noriko K. Goldsmith,* Alejandro A. Romero,* Ai-Ling Zhang,* Erick J. Morris*,1 and Lindsey Grandison² Departments of *Pharmacology, and ²Physiology and Biophysics, UMDNJ-Robert Wood Johnson Medical School, Piscataway, New Jersey, USA
Abstract Cytosine arabinoside (AraC) is a nucleoside analog that produces signi®cant neurotoxicity in cancer patients. The mechanism by which AraC causes neuronal death is a matter of some debate because the conventional understanding of AraC toxicity requires incorporation into newly synthesized DNA. Here we demonstrate that AraC-induced apoptosis of cultured cerebral cortical neurons is mediated by oxidative stress. AraC-induced cell death was reduced by treatment with several different free-radical scavengers (N-acetyl-Lcysteine, dipyridamole, uric acid, and vitamin E) and was increased following depletion of cellular glutathione stores. AraC induced the formation of reactive oxygen species in neurons as measured by an increase in the ¯uorescence of
the dye 5-(6)-carboxy-2 0 ,7 0 -dichlorodihydro¯uorescein diacetate. AraC produced DNA single-strand breaks as measured by single-cell gel electrophoresis and the level of DNA strand breakage was reduced by treatment with the free radical scavengers. These data support a model in which AraC induces neuronal apoptosis by provoking the generation of reactive oxygen species, causing oxidative DNA damage and initiating the p53-dependent apoptotic program. These observations suggest the use of antioxidant therapies to reduce neurotoxicity in AraC chemotherapeutic regimens. Keywords: antioxidant, cell death, comet, DNA strand break, ¯ow cytometry, neuroprotection. J. Neurochem. (2001) 78, 265±275.
The anticancer agent cytosine arabinoside (AraC) is one of primary chemotherapeutic agents in acute leukemia (Capizzi 1996). Use of high-dose intravenous AraC is hampered, however, by a signi®cant toxicity, particularly to the cerebellum and the cerebral cortex (Baker et al. 1991). AraC ef®cacy has been related to the phosphorylation of AraC by deoxycytidine kinase and incorporation of AraCTP into DNA during DNA synthesis, forcing the premature termination of DNA elongation, thus producing DNA strand breakage and cell death (Kufe et al. 1980). However, because terminally differentiated neurons are not synthesizing DNA, this mechanism cannot explain neurotoxicity. The neurotoxicity of AraC has been investigated most carefully in cell culture models, where AraC is shown to kill several different kinds of neurons. This observation was ®rst made by Wallace and Johnson (1989) using dorsal root ganglion neurons, but it has been since extended to several other neuronal cell types (Martin et al. 1990; Tomkins et al. 1994; Dessi et al. 1995; Sanz-Rodriguez et al. 1997; Park et al. 1998b). In each of these cases, AraC produces an apoptotic cell death which can be abrogated by any of
several different anti-apoptotic agents (Courtney and Coffey 1999). Several different mechanisms of action have been proposed for AraC (Grant 1998), though the mechanism of neurotoxicity remains obscure. The early suggestion that AraC could interfere with trophic support of neurons (Martin et al. 1990) has not been supported by later
Received February 8, 2001; revised manuscript received April 11, 2001; accepted April 11, 2001. Address correspondence and reprint requests to H. M. Geller, Department of Pharmacology, UMDNJ-Robert Wood Johnson Medical School, 675 Hoes Lane, Piscataway, NJ 08854, USA. E-mail:
[email protected] 1 Present address: E. J. Morris, Laboratory of Molecular Oncology, MGH Cancer Center, Charlestown, MA 02129, USA. Abbreviations used: AraC, cytosine arabinoside; DCFH-DA, 5-(6)carboxy-2 0 ,7 0 -dichlorodihydro¯uorescein diacetate; GPX, glutathione peroxidase; l-BSO, l-buthione sulfoximine; l-NAC, N-acetyl-lcysteine; MTT, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide; ROS, reactive oxygen species; SCGE, single-cell gel electrophoresis.
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investigations (Anderson and Tolkovsky 1999). A role for genotoxic stress as a result of AraC is supported by the observation that AraC toxicity is reduced in animals that carry a deletion of p53 (Enokido et al. 1996; Anderson and Tolkovsky 1999), a key signal of genotoxic stress (Kastan et al. 1991). Furthermore, inhibition of cyclin-dependent kinases, which lie downstream of p53 signaling, protects against AraC toxicity (Park et al. 1998a; Courtney and Coffey 1999). If AraC causes genotoxic stress, it must be through a mechanism unrelated to its direct effects on DNA synthesis. Reactive oxygen species (ROS) are known to cause DNA damage in many different cell types (Cadet et al. 1997). It has recently been reported that the drug cisplatin, which generates oxygen radicals (Masuda et al. 1994), causes a p53-dependent apoptotic cell death of neurons (Park et al. 2000). We therefore hypothesized that AraC increases production of ROS that causes both DNA damage and neuronal apoptosis. We now report that AraC causes ROS production in advance of DNA strand breakage and that DNA strand breakage and cell death can both be reduced by several different free radical scavengers; thus, the neurotoxicity of AraC can be attributed to the early generation of ROS in neurons, which produces oxidative DNA damage and neuronal apoptosis. Moreover, this observation suggests that antioxidant therapy could prevent neurotoxicity in AraC therapeutic regimens.
Materials and methods Cortical neuron culture and survival assay Neurons were cultured from the cerebral cortex of embryonic day 17 rats as previously described (Morris and Geller 1996). After dissociation, neurons were plated into multiwell dishes (approximately 100 000 cells/cm2) coated with poly-l-lysine in serum-free Neurobasal medium (Life Technologies, Gaithersburg, MD, USA) with N2 supplements (Bottenstein and Sato 1979). The purity of the cultures was assessed by demonstrating that . 99% of the cells were immunoreactive for neuro®lament, and that , 1% of the cells were immunoreactive for glial ®laments (data not shown). One day after plating, the medium was exchanged for medium supplemented with test substances where appropriate. At the times indicated in the text, neuronal viability was evaluated using a modi®cation of the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) reduction assay (Mosmann 1983). For this assay, a sterile solution of 0.1 mg/mL MTT in Neurobasal medium was added to each well of a multiwell plate, and allowed to equilibrate for 2 h. At that time, the cells were triturated and transferred to a 1.5-mL microcentrifuge tube and centrifuged at 12 000 g for 5 min at 48C. The pellet was then resuspended and solubilized in a solution of 0.8 N HCl and 1% Triton X-100 in isopropanol, the solution transferred to one well of a 96-well plate and transmission evaluated at 570 nm. Adding AraC directly to neurons at the initiation of the MTT assay did not alter MTT conversion (data not shown). All experimental points are expressed as a percentage of
reaction product in cells plated on day 0 and are reported as mean ^ SEM (n 3). Time lapse microscopy Dissociated cerebral cortical neurons were plated at a density of 2 104 cells/cm2 into 35-mm glass-bottomed microwell dishes (MatTek, Ashland, MA, USA) coated with polyethyleneimine (0.2 mg/mL) in serum-free Neurobasal medium with N2 supplements. After 24 h, cultures were placed on the stage of a Nikon Inverted microscope equipped with a custom-designed heater and incubator to maintain cultures in 5% CO2 in air at 378C and 100% humidity. Images of selected ®elds were captured every 5 min using a Dage-MTI CCD camera and stored on disk for further processing. Certain cultures were treated with AraC (30 mm) to initiate apoptosis. Measurement of [3H]AraC uptake Primary cultures of cortical neurons were exposed to [5-3H]AraC (Speci®c Activity 28 Ci/mmole; Amersham] in the absence or presence of the antioxidants dipyridamole, l-NAC, uric acid or vitamin E. One day after plating, cultures of cerebral cortical neurons (1.5 10 6 cells per 35-mm dish) were preincubated for 1 h with the antioxidant and then incubated with 3 mCi [3H]AraC and 10 mm AraC for 4 h. At the end of the incubation the media was removed and the cells were washed three times with ice-cold phosphate-buffered saline (PBS). For estimation of incorporation into DNA, 4 106 cells per 35-mm dish were incubated with 15 mCi of [3H]AraC for 24 h. In some experiments, cultures were preincubated for 1 h with 10 mm l-NAC before addition of [3H]AraC. Cells were washed three times with ice-cold PBS and subjected to phenol/chloroform extraction. DNA was precipitated from the aqueous phase with 0.3 m NaAcetate and 2.5 vol. of ethanol. Incorporation was then measured by liquid scintillation counting. Each measurement was performed in triplicate. Measurement of ROS production ROS production was measured using ¯ow cytometry according to a method adapted from Sureda et al. (1999). After dissociation, neurons were plated at a density of approximately 2.5 104 cells/ cm2 into 35-mm culture plates coated with poly-l-lysine in serumfree Neurobasal medium with N2 supplements, 0.5 mm glutamine, and antibiotics. Certain cultures were also treated with 10 mm l-buthione sulfoximine (l-BSO; Sigma, St Louis, MO, USA) at the time of plating to deplete neuronal glutathione (Zeevalk et al. 1998). 20±24 h after plating, the medium was changed to phenol red-free Dulbecco's modi®ed Eagle medium (DMEM) containing 100 mm 5-(6)-carboxy-2 0 ,7 0 -dichlorodihydro¯uorescein diacetate (DCFH-DA; Molecular Probes, Eugene, OR, USA). Cultures were then returned to the incubator for 1±2 h to allow accumulation of DCFH. AraC or H2O2 was added to cultures at the same time as DCFH for 1±2 h time points or later for shorter time points. One to 2 h after DCFH addition, neurons were gently scraped off, triturated, and ®ltered through a nylon mesh to create a cell suspension. Cellular ¯uorescence from a sample of 15 000 cells was analyzed using an Coulter EPICS Pro®le II Flow cytometer (Coulter Electronics, Miami, FL, USA). Fluorescence was excited at 488 nm and was detected using a 525 ^ 20 nm band pass ®lter. Cellular debris was excluded from analysis by gating based on forward angle light scatter. Histograms were analyzed using EPICS Workstation Software (version 4).
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Single cell gel electrophoresis Single cell gel electrophoresis (SCGE) was performed as previously described (Morris et al. 1999). Cultured neurons were harvested at the times indicated in the Results section, and ultimately resuspended at a density of approximately 5 104 cells/mL in 0.7% low-melting point agarose diluted in 0.1 m PBS and maintained at 378C. A total of 2500 cells per condition were then applied atop a very thin agarose layer applied to fully frosted glass slides by the rapid application of 150 mL of liqui®ed 0.7% agarose (SeaKem Goldw; BioWhittaker Molecular Applications, Rockland, ME, USA). The cells were then lysed by the addition of a freshly made alkaline lysis solution (1% N-lauryl sarcosine, 1% Triton X-100, 2.5 m NaCl, 100 mm EDTA, 10 mm Tris-base and 1 N NaOH; pH , 10.5) for 1 h. After lysis, the cells were then subjected to a DNA unwinding solution (0.3 m NaOH and 1 mm EDTA; pH , 11.5) for 20 min. Following unwinding, the slides were subjected to electrophoresis at 1.7 V/cm for 10 min (DNA Sub Celle gel electrophoresis apparatus; Bio-Rad, Hercules, CA, USA) using the above unwinding solution as the gel-running buffer. After electrophoresis, the slides were then stained with SybrGoldw (Molecular Probes). Nuclei were visualized using epi¯uoresent illumination on a Zeiss Axioplan microscope. Under these conditions, the amount of migrating, broken DNA is a function of both the number of DNA strand breaks and the size of the strands. The broken DNA can be visualized as a bright migrating `tail' observed emanating from the nucleus forming a structure very reminiscent of a `comet'; hence the descriptive `comet-assay'. Images of nuclei in random, non-overlapping ®elds were captured using an MTI CCD-300 camera and a Macintosh Quadra 700 with a Scion LG-3 frame grabber board. Images were then analyzed with the NIH Image program (v. 1.61, available at http://rsbweb.nih. gov/nih-image/index.html) and further processed with Adobe Photoshop software. DNA damage was quanti®ed by determining the tail moment, a function of both the tail length and intensity of DNA in the tail (Hellman et al. 1995).
Results AraC produces an apoptotic cell death in dissociated embryonic cerebral cortical neurons AraC has been reported to be toxic to several types of cultured neurons, including sympathetic ganglion neurons (Wallace & Johnson 1989; Tomkins et al. 1994), cerebellar granule neurons (Dessi et al. 1995; Enokido et al. 1996), as well as PC12 cells (Park et al. 1998a). Because AraC is frequently used to kill non-neuronal cells in culture, we cultured dissociated neurons in serum-free conditions designed to suppress cell proliferation and evaluated the effects of AraC on neuronal survival 24 h later using the MTT assay (Fig. 1). As has been reported for cerebellar granule neurons (Courtney and Coffey 1999), the IC50 for AraC toxicity to dissociated cerebral cortical neurons increased with the duration of time in culture. We found an IC50 of about 3 mm for 24 h of treatment (Fig. 1) beginning at time of culture. Because it has been reported that MTT reduction may not accurately re¯ect cell death
Fig. 1 AraC causes a concentration-dependent cell death in cerebral cortical neurons. Dissociated embryonic cerebral cortical neurons were maintained in serum-free N2±Neurobasal medium for 24 h after plating. The indicated concentratios of AraC were added at 24 h. Neuronal survival were evaluated after 24 h later using the MTT assay. Neuronal survival was graphed as the percentage of survival in untreated cultures (mean ^ SD).
(Abe and Saito 1999), certain experiments were conducted using both MTT and CFDA staining for vital neurons, which gives an accurate indication of neuronal cell death (Petroski and Geller 1994); no differences in results were found using these two methods (data not shown). Therefore, all further studies of neurotoxicity were conducted using the MTT assay at day 1 in vitro. AraC has been reported to cause an apoptotic death in neurons (Wallace & Johnson 1989; Tomkins et al. 1994; Dessi et al. 1995; Enokido et al. 1996). We therefore examined the response to AraC treatment using time lapse microscopy (Morris and Geller 1996). While untreated neurons remained healthy in the chamber, dissociated embryonic cerebral cortical neurons treated with AraC displayed the typical morphological features of apoptosis, including zeiosis and the formation of apoptotic bodies (Fig. 2d). After 24 h, cultures were ®xed and stained with DAPI to evaluate the the appearance of apoptotic nuclei using the morphological criteria of condensed chromatin (Morris and Geller 1996). This induction of chromatin condensation was time- and concentration-dependent (Fig. 2e). The potency of AraC for producing apoptosis varies depending upon the tissue culture substrate, the plating density of the neurons and the culture medium (data not shown). However, using the culture conditions speci®ed in the Materials and methods section, we found an IC50 for production of apoptotic nuclei of approximately 10 mm at 24 h of exposure to AraC at day 1 in vitro (Fig. 2e).
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Fig. 2 AraC causes apoptotic death in cultured cerebral cortical neurons. Dissociated embryonic cerebral cortical neurons were maintained in serum-free N2±Neurobasal medium for 24 h after plating. AraC was added at 24 h. (a and b) Representative time-lapse images of neurons obtained (a) at the time of placement into the culture chamber and (b) 20 h later showing no cell death during the observation period. Note that some neurons move and extend processes over this period of time. Images of neurons at the time of addition of 30 mM AraC (c) and 20 h after AraC addition (d) demonstrating fragmented neuronal processes and apoptotic bodies (arrows). Not all neurons proceed at the same time, as some neurons are in the early stages of zeiosis (arrowhead). Bar 20 mM. (e) AraC-induced apoptosis is concentration- and time-dependent. Dissociated embryonic cerebral cortical neurons were maintained in serum-free N2±Neurobasal medium. Cultures were removed, ®xed and reacted with DAPI at the indicated time points. The percentage of apoptotic nuclei in control and AraC-treated cultures were counted from images.
Antioxidants attenuate AraC toxicity To evaluate if ROS could be involved in the neurotoxicity of AraC, cultured cerebral cortical neurons were treated with AraC (1±30 mm) either alone or in combination with the antioxidant compounds N-acetyl-l-cysteine (l-NAC, 1±30 mm) (Vanderbist et al. 1996), dipyridamole
Fig. 3 Antioxidants attenuate AraC-induced neuronal death. Dissociated embryonic cerebral cortical neurons were maintained in serum-free N2±Neurobasal medium for 24 h after plating. Antioxidants were added to the medium 1 h prior to AraC exposure. Neuronal survival was measured 24 h later using the MTT assay. Neuronal survival was graphed as the percentage of survival (mean ^ SD) as compared to control neurons treated only with the antioxidant. Each of the antioxidants signi®cantly reduced neuronal death as compared to cells treated with AraC alone ( p , 0.01, twoway ANOVA). (a) N-acetyl-L-cysteine; (b) Uric acid (n 3 per condition); (c) Dipyridamole; (d) Vitamin E. X, Control; W, 1 mM; P, 3 mM; L, 30 mM.
(1±30 mm) (Iuliano et al. 1995), vitamin E (100±500 mg/ mL) (van Acker et al. 1993), uric acid (1±30 mm) (Singh et al. 1998), and vitamin C (1±30 mm) (Cathcart 1985). Neuronal survival was measured after 24 h of exposure using the MTT assay. As has been reported for other neuronal types (Martin and Wiley 1995), we found that treatment with the antioxidants, l-NAC, dipyridamole, uric acid, and vitamin E increased the survival of cultured cerebral cortical neurons grown in serum-free medium containing N2 by 20±40%. Therefore, the dose±response curves for alteration in AraC-induced cell death were normalized to survival in the absence of AraC. l-NAC, uric acid, dipyridamole and vitamin E each produced a dose-related increase in the survival of cultured cerebral cortical neurons in response to AraC (Fig. 3), while cotreatment with vitamin C produced inconsistent effects, with protection being observed only in some experiments (data not shown). The antioxidants each produced a concentration-dependent shift in the dose±response curve for AraC-induced neurotoxicity. The protection for all
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Fig. 4 AraC toxicity is reduced by 2 0 -deoxycytidine (2'dc). Dissociated embryonic cerebral cortical. neurons were maintained in serum-free N2±Neurobasal medium for 24 h after plating. AraC was added to the cultures either alone or in combination with 2 0 -deoxycytidine. Neuronal survival was measured 24 h later using the MTT assay. Neuronal survival was graphed as the percentage of survival (mean ^ SD) as compared to untreated neurons. 2 0 -deoxycitidine signi®cantly increased neuronal survival as compared to cultures treated with AraC alone ( p , 0.01, one-way ANOVA).
agents was observed at all concentrations of AraC. However, the maximal protection was observed at the highest concentration of AraC used (30 mm), where the antioxidants increased survival by 200±300% as compared to untreated cultures. The most effective agents were l-NAC and uric acid, which allowed for a 300% increase in the survival of untreated cultures, while the highest concentrations of dipyridamole and vitamin E increased survival by 200%. Thus, antioxidants protect neurons against AraC-induced cell death. AraC neurotoxicity is dependent upon uptake and phosphorylation of AraC AraC is taken up into cells via several different nucleoside transporters (Wiley et al. 1983) and phosphorylated to Table 1 Effects of antioxidants on AraC accumulation Additon
Percentage uptake
None Dipyridamole (1 mM) Dipyridamole (10 mM) L-NAC (10 mM) Uric acid (10 mM) Vitamin E (500 mg/mL)
100 111 50.9 92 73.6 77.8
^ ^ ^ ^ ^ ^
6.1 3.2 4.1* 4.3 3.3* 6.2*
Cerebral cortical neurons were dissociated and plated into 35-mm dishes for 24 h. Neurons were then pre-incubated for 1 h with the antioxidant, at which time [3H]AraC was added for an additional 4 h. *Signi®cantly different from control, p , 0.05, t-test.
AraCTP (Schrecker 1970). Toxicity to dividing cells (as measured by clonogenic survival) has been correlated with uptake of AraCTP into DNA and inhibition of DNA polymerase a (Yoshida et al. 1977; Kufe et al. 1980). In these dividing cells, AraCTP reduces DNA chain initiation and elongation while promoting chain termination. Since the cultured neurons are postmitotic and not dividing, and since quiescent neurons do not express DNA polymerase a (Waser et al. 1979), it is unlikely that AraC toxicity is through inhibition of DNA replication. To con®rm this, we measured the uptake of [3H]AraC into the cytoplasm and nuclei of cortical neurons in culture. Incubation of [3H]AraC with cortical neurons indicated an initial rapid uptake into the cells followed by a prolonged slower uptake process (data not shown). Over a 4-h incubation period, we found that neuronal uptake was 72 ^ 4.4 pmol AraC/106 cells. These observations are consistent with previous characterization in non-neuronal cells of a low af®nity, high capacity transport of AraC into the cell through nucleoside transporters (Wang et al. 1997) and a trapping within the cell by a high af®nity, low capacity phosphorylation by deoxycytidine kinase (Wiley et al. 1985). We found a very small amount of AraC in the nuclear fraction, estimated to be 4.1 ^ 0.42 fmol AraC/106 cells/24 h, barely above background. This nuclear accumulation of AraC was less than 0.1% of the AraC accumulated into cultured cortical neurons. AraC-induced neuronal cell death has been reported to be inhibited by 2 0 -deoxycytidine (Wallace & Johnson 1989; Dessi et al. 1995), a competitive inhibitor of both uptake into the cell and the phosphorylation of AraC (Balzarini et al. 1987). We therefore evaluated whether 2 0 -deoxycytidine could inhibit AraC-induced cell death of cerebral cortical neurons. AraC (100 mm) was added to cerebral cortical neurons alone or in combination with increasing concentrations of 2 0 -deoxycytidine (0.1±10 mm). As can be seen in Fig. 4, 2 0 -deoxycytidine produced a dose-related decrease in AraC neurotoxicity. This indicates that the toxic actions of AraC require uptake into cells and/or phosphorylation. Since antioxidants were observed to reduce AraC-induced neurotoxicity, we examined their effect on AraC uptake as a potential mechanism. The uptake of [3H]AraC into cultured cerebral cortical neurons was measured in the absence or presence of the antioxidants, which were found to be protective (Table 1). While dipyridamole is known to reduce nucleoside uptake (Paterson et al. 1980), the other antioxidants used have not been reported to interfere with nucleoside transport. We found that dipyridamole caused a concentration-related decrease in AraC uptake, with no inhibition being observed at 1 mm, and a signi®cant inhibition being observed at 10 mm. Uric acid and vitamin E had a smaller, but signi®cant, effect. Since AraC is phosphorylated and retained inside the cell, these reductions in initial velocity of uptake may not represent alterations in
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Fig. 5 AraC toxicity is enhanced by glutathione depletion. Cerebral cortical neurons were dissociated and maintained in serum-free N2±Neurobasal medium with (gray bars) or without (black bars) 15 mM BSO for 24 h after plating. AraC was added in the indicated concentrations and neuronal survival measured 24 h later using the MTT assay. Neuronal survival was graphed as the percentage of survival (mean ^ SD) as compared to untreated neurons. Survival in BSO-treated cultures was signi®cantly less than control cultures ( p , 0.001, two-way ANOVA).
®nal intracellular concentration during the 24-h treatment period. AraC toxicity is potentiated by glutathione depletion Protection against ROS is afforded by several different systems. The main antioxidant defense system consists of a series of enzymes which include superoxide dismutase,
Fig. 6 AraC and H2O2 induce ROS production in cultured cerebral cortical neurons. Dissociated embryonic neurons were maintained in 35-mm culture dishes in serum-free N2±Neurobasal medium for 24 h containing 10 mM BSO. After 20±24 h, neurons were loaded with the ROS indicator dye DCFH-DA and treated with either AraC or H2O2. Neurons were then dissociated 1 h later and DCFH ¯uorescence was measured using ¯ow cytometry in a sample of 15 000 neurons. This histogram represents DCFH ¯uorescence in dissociated neurons exposed to saline (solid line), 10 mM H2O2 (solid line) or 30 mM AraC (dotted line). An Overton analysis of the two histograms revealed that the 39.1% of the cells increased their ¯uorescence intensity in response to 10 mM H2O2, while 37.1% of cells increased their ¯uorescence intensity in response to 30 mM AraC.
Fig. 7 AraC induces DNA strand breaks in cerebral cortical neurons. Embryonic cerebral cortical neurons were maintained in serum-free N2±Neurobasal medium for 24 h after plating. Cells were treated for 4 h in the indicated conditions and then harvested and subject to single-cell gel electrophoresis. DNA was stained with Sybr-Gold and images were captured of individual nuclei. (a) Control; (b) AraC (10 mM); (c) AraC (10 mM) 1 L-NAC (1 mM); (d) L NAC (1 mM). Bar 25 mM.
catalase, and glutathione peroxidase (GPX). These enzymes act in concert to protect cells from oxidative stress (Michiels et al. 1994). Cerebral cortical cultures were treated with 10 mm BSO at the time of plating. This concentration of concentration of BSO depletes over 60% of glutathione from cultured neurons in 24 h (Zeevalk et al. 1997), yet has a minimal effect on neuronal survival during this period. AraC was added 24 h later, and the survival of neurons was measured at 24 h after AraC addition. Figure 5 presents the results of a typical experiment showing that glutathione depletion with BSO signi®cantly ( p , 0.01, two-way anova) increased the toxicity of AraC. Thus, reducing the ability to defend against ROS by depletion of glutathione enhances AraC toxicity. AraC causes ROS production Measurement of ROS production was accomplished by the ¯ow cytometric detection of the ¯uorescence of the dye DCFH (Grierson et al. 1992; Sureda et al. 1999). Cultures of embryonic cerebral cortical neurons were prepared and loaded with DCFH-DA as speci®ed in Materials and methods. AraC was added to the neuronal suspension and samples were taken for ¯ow cytometric analysis at intervals between 15 min and 2 h. Cellular ¯uorescence was then measured in a sample of 15 000 cells. In cultures treated with 10 mm BSO, AraC (30±100 mm) induced a measurable increase in DCFH ¯uorescence. This increase was comparable to that obtained by treating neurons with 10 mm H2O2. A typical experment is illustrated in Fig. 6, which demonstrates that treatment of cultures with either 30 mm AraC or 10 mm H2O2 produced a rightward shift in the ¯uorescence histogram taken at 1 h after
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This difference may be related to the rate of accumulation of AraC as demonstrated above.
Fig. 8 AraC-induced DNA strand breaks are dose- and timedependent. Dissociated embryonic cerebral cortical neurons were maintained in serum-free N2±Neurobasal medium for 24 h after plating. Cells were then treated with AraC [10 (solid bars) and 30 mM (hatched bars) for the indicated times and then harvested and subject to single-cell gel electrophoresis. Images were obtained as in Fig. 6 and tail moments were calculated for a sample of 100 cells for each condition. The effects of AraC are both concentration- and time-dependent ( p , 0.01, one-way ANOVA). Results are presented as mean ^ SEM.
treatment. The percentage of cells that shifted their ¯uorescence in response to H2O2 and AraC were determined using an Overton analysis, which is a cumulative subtractive technique. This showed a shift in ¯uorescence in 39% of the cells in response to 10 mm H2O2 and 37% in response to 30 mm AraC. While the production of ROS in response to 30 mm AraC was observed only after 1 h of AraC treatment, we found that 100 mm AraC produced a measurable shift in ¯uorescence at 15 min after treatment (data not shown).
AraC produces DNA strand breaks AraC-induced neurotoxicity is p53-dependent (Enokido et al. 1996). p53 is normally involved in the signaling of DNA strand breaks following toxic insults in many different cell types, including neurons (Morris and Geller 1996). This suggests that ROS production by AraC could cause DNA strand breakage. We therefore measured the production of DNA single-strand breaks by AraC using single cell gel electrophoresis (the `comet' assay) (Morris et al. 1999). Figure 7 demonstrates images of single neurons stained with the DNA stain Sybr-Gold. In the control conditions (Fig. 7a), there was a very low level of DNA strand breaks as shown by the absence of a visible comet tail. Treatment with AraC caused signi®cant DNA strand breakage, manifested by the formation of comet tails in all the treated neurons (Fig. 7b). The relative level of DNA strand breakage in terms of the comet moment (Morris et al. 1999) was computed from images of comets from treated cells and normalized to the levels in untreated cells. While all of the neurons showed DNA strand breakage in response to AraC, the extent of DNA damage by AraC is both concentration- and time-dependent (Fig. 8). Signi®cant DNA damage was apparent at 2 h after AraC treatment, at which time a doubling of the comet moment was observed after 10 mm AraC, and a sixfold increase was observed after 30 mm AraC. Comet moments continued to increase in size with the duration of the treatment. Thus, AraC induces DNA strand breaks in cultured cerebral cortical neurons, and these strand breaks are observed to occur following ROS production, but before any morphological signs of apoptosis.
Fig. 9 AraC-induced DNA strand breaks are reduced by antioxidants. Dissociated embryonic cerebral cortical neurons were maintained in serum-free N2±Neurobasal medium for 24 h after plating. Cells were then treated with 10 mM AraC either alone or in combination with (a) L-NAC, (b) uric acid, (c) dipyridamole or (d) vitamin E for 4 h and then harvested and subject to single-cell gel electrophoresis. Images were obtained as in Fig. 4 and tail moments were calculated for a sample of 100 cells for each condition. Results are presented as mean ^ SEM. Each of the antioxidants signi®cantly reduced comet formation in response to AraC ( p , 0.01, one-way ANOVA). Both dipyridamole and vitamin E induced signi®cant comet formation (*p , 0.01 as compared to control, Student's t-test).
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Antioxidants attenuate DNA strand breakage in response to AraC DNA strand breakage is one of the major effects of ROS (Cadet et al. 1997). We therefore evaluated if the antioxidants that afforded protection from cell death could reduce the formation of DNA single-strand breaks as measured by SSGE. To do this, cultures of cerebral cortical neurons were treated with either AraC, antioxidants, or a combination of the two. The antioxidants l-NAC (Fig. 7c), uric acid, dipyridamole, and vitamin E each signi®cantly reduced the level of DNA strand breaks in response to AraC (Fig. 9). The results with vitamin C were highly variable, with reduction of DNA strand breakage observed in only a minority of experiments (data not shown). High concentrations of dipyridamole and vitamin E produced signi®cant strand breakage in the absence of AraC. This was not observed for the other antioxidants used. It should be noted that the antioxidant agents that produce DNA strand breaks were also less ef®cacious in neuroprotection assays (Fig. 3). Discussion Our results show that AraC neurotoxicity results from ROS generation. The generated ROS is then causally related to apoptosis. Scavenging of ROS protected against AraCinduced cell death, and toxicity was also enhanced by reducing antioxidant defenses. AraC also produced DNA single-strand breaks in neurons and antioxidants reduced DNA strand breaks in parallel with reducing neurotoxicity. We propose the model illustrated in Fig. 10, which includes a sequence of events linking AraC to ROS production, DNA damage and neurotoxicity. AraC has been found to be neurotoxic for many different types of neurons in culture (Wallace & Johnson 1989; Martin et al. 1990; Tomkins et al. 1994; Dessi et al. 1995; Sanz-Rodriguez et al. 1997; Park et al. 1998b). In replicating cells, AraC is toxic following its incorporation into DNA with the resultant inhibition of DNA polymerase activity and chain termination (Kufe and Major 1982). However, terminally differentiated neurons do not actively synthesize DNA, and we have shown here that there is no appreciable AraC incorporation into DNA of cultured neurons. Thus it is unlikely that AraC induces DNA damage and apoptosis by inhibition of DNA synthesis. Furthermore, the potency of AraC in inducing cell death is reduced as granule neurons mature in culture, and this reduction in cell death is correlated with an increase in DNA repair capability (A. Romero and H. Geller, unpublished observations). AraC produces an apoptotic neuronal death, which is consistent with all previous studies of AraC neurotoxicity. ROS produces either an apoptotic or necrotic cell death in many different cell types, depending upon the level of cellular stress: low levels of stress cause apoptosis, while
Fig. 10 Proposed pathway of AraC-induced neurotoxicity. AraC is taken up into cells where it becomes phosphorylated to AraCTP. AraCTP then induces the production of ROS which causes oxidative DNA strand breaks and initiates the p53-dependent apoptotic process. Cerebral cortical neurons were dissociated and plated into 35mm dishes for 24 h. Neurons were then preincubated for 1 h with the antioxidant at which time [3H]AraC was added for an additional 4 h. * signi®cantly different from control, p , 0.05, t-test.
higher levels of stress produce necrotic cell death (McConkey 1998). ROS produces DNA strand breakage (Cadet et al. 1999). It may be that low levels of ROS and DNA strand breakage, as are produced following AraC exposure, serve to trigger apoptosis by activating redoxdependent signaling pathways (Finkel 2000), such as the Ref-1 protein, which signals to p53 (Jayaraman et al. 1997), while higher levels of ROS cause more severe effects on cellular protein and lipid functions, causing necrosis. The fact that AraC-induced toxicity and DNA damage are both reduced by free radical scavengers, and that AraC apoptosis is converted to necrosis by additional oxidative stress (Shacter et al. 2000) supports the idea that the level of oxidative stress determines whether a cell dies via apoptosis or necrosis. The evidence for DNA strand breakage in response to AraC was obtained using the comet assay, a sensitive method for detecting single-strand breaks in nuclear DNA (Fairbairn et al. 1995). The question arises as to whether the DNA single-strand breaks detected by the comet assay could have been due to DNA fragmentation that occurs in all apoptotic nuclei. This is not likely for two reasons. The ®rst is that AraC-induced comet formation in all neurons by 2 h after treatment, a time when only a small percentage of nuclei showed any sign of chromatin condensation. This time course has also been reported for AraC-induced apoptosis in HL-60 leukemia cells, where over expression of Bcl-2 was able to prevent apoptosis and apoptotic DNA fragmentation, but not DNA strand breakage (Bullock et al.
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1996). Secondly, images of comets were obtained only from cells that displayed intact nuclei after staining with the Sybr-Green DNA stain (Fig. 4). In fact, we have never observed fragmented nuclei after SSGE, even in the presence of massive apoptotic death (data not shown). This is likely due to the fact that dead cells and apoptotic bodies are lost during the initial suspension procedure that detaches neurons from the substrate (data not shown), and thus our measurements of DNA strand breaks are obtained from cells that have not progressed into the stage of chromatin condensation. Thus, these measurements are taken as evidence of an early DNA strand breakage before the onset of apoptosis. The ability of ROS to cause both DNA damage and cell death is well established. In addition, DNA damage is well established to lead to cell death. Our observations support ROS generation and the subsequent DNA damage as the mechanism for AraC-induced neurotoxicity. However, AraC has been reported to produce other cellular responses. AraCinduced increases in the activity of stress-activated protein kinases, including Jun kinases (Jarvis et al. 1998) and ceramide (Strum et al. 1994) have been related to promotion of apoptosis, while increases in protein kinase C (Kharbanda et al. 1994) and MAP kinases (Jarvis et al. 1998) are thought to be anti-apoptotic. It is not likely that the stress-activated protein kinase pathway is involved in neuronal apoptosis, because AraC did not increase Jun kinase phosphorylation in neurons (Anderson and Tolkovsky 1999) and blocking Jun kinase/p38 activation had no effect on AraC-induced neuronal apoptosis (Courtney and Coffey 1999). AraCinduced ceramide production (Strum et al. 1994) is likely mediated by the stress-activated protein kinase (Jarvis et al. 1998), although oxidative stress has been reported to directly activate neutral sphingomyelinase and ceramide production without the necessity for DNA damage (Goldkorn et al. 1998). However, inhibition of sphingomyelinase activity did not inhibit AraC-induced cell death (Jarvis et al. 1998), indicating that ceramide is unlikely to be a downstream effector for AraC in apoptotic signaling. Unresolved is the mechanism by which AraC causes ROS generation. It appears that AraC-induced ROS is an early event based on the time course of its appearance. ROS production occurs in advance of DNA damage since antioxidants prevent DNA strand breakage. AraC has been reported to increase ATP production and enhance the activity of cytochrome c oxidase in leukemia cells (Van den Muus et al. 1991), which might lead to ROS production. However, we have not observed any difference in ATP levels between AraC-treated and untreated neurons (N. K. Goldsmith, unpublished observations). Furthermore, an effect of AraC on mitochondrial DNA, which could alter mitochondrial function, is not likely because AraC appears to be selectively incorporated into nuclear, rather than mitochondrial, DNA (Zhu et al. 2000). On the other hand,
AraC could reduce the levels of antioxidant defenses, such as the level of glutathione. However, the fact that AraC increased DCFH ¯uorescence only after depletion of glutathione (Zeevalk et al. 1998) would suggest that glutathione levels are not altered in response to acute AraC treatment. Alternatively, AraC may lead to ROS generation as a result of its effects on lipid metabolism. AraC monophosphate can be utilized by cholinephosphotransferase to form diradylglycerol and AraCDP choline (Kucera and Capizzi 1992). While the diradylglycerol activates protein kinase C (PKC), potentially it could also be de-esteri®ed by diglyceride lipase to yield fatty acids such as arachidonic acid (Prescott and Majerus 1983). The subsequent metabolism of these fatty acids might then yield ROS. Thus, the exact mechanism by which AraC produces ROS in neurons remains to be determined. AraC neurotoxicity has been well-recognized as a consequence of therapy for acute leukemias. We have presented evidence for the early signaling pathway for neuronal cell death in response to AraC: AraC causes an increase in ROS generation, oxidative DNA damage, and p53-dependent apoptosis. This is a novel signal transduction pathway for production of apoptosis by AraC. While AraC neurotoxicity is a potential problem after high-dose intravenous infusion regimens (Baker et al. 1991), where calculations from published data (Burk et al. 1997; Groothuis et al. 2000) suggest that brain concentrations can be in the micromolar range, toxicity is likely to be much more signi®cant following intrathecal administration of AraC, where concentrations are several thousand times higher (Groothuis et al. 2000). Because antioxidants reduce the neurotoxic actions of AraC, it is possible that treatment with antioxidants may have a neuroprotective action in clinical situations employing AraC. Acknowledgements The authors wish to thank the members of the HMG lab for their constant support. Ms Tina DeCoste for her assistance with ¯ow cytometry and Lily Moy for her assistance with measurements of ATP and glutathione. This work was supported by the National Institutes of Health (R01 NS 36443 and P01 ES10874).
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