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Cytosine arabinoside is neurotoxic to chick embryo spinal cord motoneurons in culture
Neuroscience Letters 223 (1997) 141–144
Cytosine arabinoside is neurotoxic to chick embryo spinal cord motoneurons in culture Cesar Sanz-Rodriguez1, Jacint Boix, Joan X. Comella* Unitat de Neurobiologia Molecular, Departament de Cie`ncies Me`diques Ba`siques, Facultat de Medicina, Universitat de Lleida, Rovira Roure 44, 25198 Lleida, Spain Received 23 December 1996; revised version received 22 January 1997; accepted 22 January 1997
Abstract Cytosine arabinoside (1-b-d-arabinofuranosylcytosine, AraC) is a commonly used antimitotic agent that kills proliferating cells by inhibiting DNA synthesis. We report that AraC is toxic to cultured chick embryo spinal cord motoneurons (MTNs) in a concentrationdependent fashion with an EC50 of about 2 mM. Interestingly, this type of MTN death is specific, resembles that occurring upon muscle extract (MEX) trophic deprivation regarding its morphological and temporal characteristics, and has apoptotic features, as judged by observation of nuclear morphology. The death of AraC-treated MTNs can be blocked by 2′-deoxycytidine (dC), a pyrimidine metabolite AraC is structurally related to. Overall, these findings suggest that dC may participate in a pathway, different from inhibition of DNA synthesis, that is necessary for cultured MTNs to respond to the trophic activities present in MEX. 1997 Elsevier Science Ireland Ltd. Keywords: Motoneurons; Muscle extract; Cytosine arabinoside; Cell death; Apoptosis; Nitrobenzylthioinosine; 2′-Deoxycytidine
Cytosine arabinoside (1-b-d-arabinofuranosylcytosine, AraC) is a pyrimidine antimetabolite structurally related to 2′-deoxycytidine (dC). It is a useful tool to selectively kill dividing cells, since it may be incorporated into DNA, thus causing inhibition of DNA synthesis [7]. Indeed, AraC is widely used in tissue culture of non-mitotic cells to eliminate proliferating cells, such as fibroblasts or glia, as well as a chemotherapeutic agent for certain lymphoproliferative disorders. Yet, AraC may also affect certain non-dividing cells. In vitro, AraC is more toxic to neurons than other antimitotic drugs [1,11]. Moreover, cancer patients treated with AraC can present with central and peripheral nervous system degeneration [6,10]. Several researchers have used in vitro models to examine the mechanisms underlying the neurotoxicity of AraC. This drug blocks specifically the survival in vitro of postmitotic parasympathetic, sympathetic, and sensory neurons stimu* Corresponding author. Tel.: +34 73 702438; fax: +34 73 702426; e-mail: [email protected] 1 Present address: Servicio de Hematologı´a, Hospital Universitario de la Princesa, 28006 Madrid, Spain.
lated by neurotrophic factors [8,12,15] as well as of cerebellar neurons [3]. The neuronal death induced by AraC is apoptotic [3,8,12], in agreement with what has been reported for other cell types [5,9]. AraC appears to exert its neurotoxic effect by interfering with a dC-dependent step included in neurotrophic factor signalling pathways, which is independent of DNA synthesis or repair [8,15]. In this study, we investigated whether AraC is also neurotoxic to postmitotic spinal cord motoneurons (MTNs). MTNs were purified from 5.5-day-old chick embryos (COPAGA, Spain) as previously reported [2]. First, spinal cord MTNs that had been cultured for 24 h in the presence of muscle extract (MEX), were treated with varying concentrations (10 nM–100 mM) of AraC while in the presence of MEX for 5 additional days. Both MTN survival and morphological changes occurring during the experiment were assessed in detail. At very high concentrations of AraC (100 mM), MTNs degenerated very rapidly while swelling and accumulating vacuoles, in what clearly looked like a dose-related and non-specific toxic effect. However, at concentrations of AraC ranging from 10 nM to 10 mM, no changes were observed in the exposed cul-
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Fig. 1. The neurotoxic effect of AraC on spinal cord MTNs is potent (EC50 ca. 2 mM) (A) and specific, and can be prevented by dC and NBTI (B). MTNs were seeded at a density of 10 000/well in 96-well culture dishes, and grown for 24 h in the presence of MEX (300 mg/ml). At that time, cells in the central area of every culture well were counted at 20 × magnification with phase-contrast inverted microscopy. Cells considered to be alive, bore neurites longer than 2 cell diameters. For each experiment, the value taken as the corrected 100% survival, represented the average of 6–8 wells. Thereafter, culture wells were washed and next exposed to fresh medium containing appropriate concentrations of the drugs. In a first series of experiments, the potency of AraC neurotoxicity was determined by exposing MTN cultures to concentrations of AraC ranging from 10 nM to 100 mM (A). In a second series of experiments (B), MTNs grown in the presence of MEX were treated with dC (1 mM) or NBTI (10 mM) in the presence of AraC (1 mM), aphidicolin (1 and 10 mM), or 5′-fluorodeoxyuridine (1 and 10 mM). Additional groups of MTNs were always maintained in MEX alone or without any trophic support (Depr). MTN survival was evaluated in the same microscopic field 5 days later. Survival was expressed as a percentage of neuronal counts with respect to the previously established 100% value. Each point represents the mean ± SEM of 6–8 wells from a single experiment that was repeated twice more with results comparable to those presented (*P , 0.05, significantly different from control, MEX-treated cultures using the Mann–Whitney U-Wilcoxon test). The different supplements and drugs used were obtained from Sigma. Tissue culture dishes and culture media were obtained from Corning and Gibco, respectively.
tures over the first 48 h. By 72 h, MTNs exposed to .1 mM AraC began to show signs of atrophy, which was followed by dramatic degeneration and condensation of the cell bodies over the following 24–48 h. The neurites became progressively thinner and discontinuous. After 5 days of AraC treatment, the culture dish was covered with degenerated, phase-dark residua of cell bodies and neurites. At concentrations of AraC ,0.5 mM, MTNs looked healthy. When incubated in concentrations of AraC between 0.5 and 1 mM, MTNs appeared somewhat atrophic and partially degenerated, even though many of them were still alive. Quantitative analysis of survival revealed an EC50 of approximately 2 mM, indicating that spinal cord MTNs were very sensitive to AraC (Fig. 1A). Cultured MTNs were also incubated in the presence of two other antimitotic drugs, aphidicolin and 5′-fluorodeoxyuridine, whose ability at killing proliferating cells is comparable to that of AraC. These agents inhibit DNA synthesis and repair, respectively, thus sharing with AraC the same general mechanism of action. None of these drugs (which were both used at 1 and 10 mM for 5 days) had any significant
neurotoxic effect on MTNs (Fig. 1B), suggesting that the cytotoxic effect of AraC was specific and not just the result of a general toxic effect since similar concentrations of AraC were clearly neurotoxic. Under phase-contrast microscopy, the morphology of AraC-treated cultured MTNs closely resembled that of MTNs deprived of MEX [2]. To compare these deaths more carefully, we compared the time courses of death caused by MEX deprivation and by AraC (1 mM) treatment (Fig. 2). Interestingly, the kinetics of death induced by AraC were similar to MEX deprivation, though delayed in onset about 48 h. During that delay period, MTNs remained apparently unaffected. In both cases, the extent of MTN death correlated with the morphological degeneration of the cells. We have previously demonstrated that the death of cultured chick embryo spinal cord MTNs upon MEX deprivation is apoptotic [2]. The close resemblance found between MTN death induced by AraC and that caused by trophic deprivation, prompted us to examine the nuclear morphology of dying AraC-treated cultures. MTNs that had been grown for 48 h in the presence of MEX, were exposed to AraC (1 mM) for varying lengths of time (from 24 to 120 h after addition of AraC). Control cultures either in the presence or in the absence of MEX were also maintained. MTNs were next fixed, stained with the apoptotic-specific Hoescht 33258 dye, and finally observed with a microscope equipped with fluorescence and UV filters. Parallel examination of the same microscopic field with Nomarsky optics allowed the precise identification and, thus, quantitation of those neurons
Fig. 2. Both MEX deprivation (filled triangles) and AraC (filled squares) cause the rapid degeneration of MTNs over a 24-h period following an initial lag in which neurons appear unaffected. MEX-treated cultures (open circles) showed a very high survival response over the culture time. The method to evaluate cell survival was the same as in Fig. 1. At times indicated on the abscissa, survival was evaluated. Survival was expressed as the percentage of neurons remaining with respect to the number of neurons present in the culture when drugs were added. Each point represents the mean ± SEM of 6–8 wells from a single experiment that was repeated twice with results comparable to those presented.
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Fig. 3. AraC-induced death of MTNs is apoptotic: (A) quantitative and (B) morphological evaluation. MTNs were cultured at a density of 150 000 cells in 35 mm culture plates. After MTNs had grown for 48 h in the presence of MEX (300 mg/ml), cultures were exposed to AraC (1 mM) (filled circles). An additional group of MTNs was always maintained in the presence of MEX (open squares), while a third group was deprived of trophic support (open circles). At 24, 48, 72, 96, and 120 h after addition of AraC, MTNs were fixed at 4°C with 2.5% (v/v) glutaraldehyde in phosphate-buffered saline (pH 7.4) for 30 min. MEX-treated cultures were processed 5 days after the establishment of the conditions and deprived cultures after 24 h. Thereafter, MTNs were stained for 1 h with 0.05 mg/ml Hoescht 33258 dye. The culture plates containing the neurons were coverslipped and counted with a vertical microscope equipped with epifluorescence and UV filters. Parallel examination of the same microscopic field with Nomarsky optics allowed the precise identification of apoptotic neurons. (A) Apoptotic MTNs were counted at 20 × magnification. The cell counts for each experimental condition consisted of 6 fields/slide and 3 slides/ condition. Values represented are the mean ± SEM of three replicates from a single experiment that was repeated twice more with results comparable to those presented. MEX-treated cultures of MTNs untreated (a) and treated (b) with AraC were viewed with epifluorescence optics after Hoescht 33258 staining. A clear increase in the number of MTNs undergoing apoptosis in response to the AraC treatment could easily be appreciated. Arrows indicate apoptotic neurons, whose DNA has condensed into brightly stained masses. Scale bar, 40 mm.
that exhibited an apoptotic morphology, i.e. those cells exhibiting micronucleation of their DNA and those that had become ghost cells due to the entire degradation of their DNA. After 4–5 days in AraC-treated cultures 10– 15% of MTNs displayed apoptotic nuclear morphologies (Fig. 3A). The nuclei appeared condensed and with a brighter fluorescence than normal nuclei, indicating that chromatin had aggregated and collapsed at the nuclear membrane. Condensation of the chromatin at the nuclear membrane was followed by fragmentation of the nucleus (Fig. 3B). It should be noted that 3–4% of MTNs were found to apoptose spontaneously in cultures exposed to MEX only, while this value increased to about 9% in MEX-deprived cultures (Fig. 3A). These results suggested that AraC-induced MTN death was most likely apoptotic. AraC neurotoxicity seems to involve a process specific for dC. This hypothesis was based upon the observation that dC specifically counters the neurotoxic effect of AraC
in several neuronal types [8,12,15]. In order to validate these results for embryonic MTNs, established cultures were treated with AraC (1 mM) in the presence of an equimolar concentration of dC. After 5 days, dC entirely blocked the neurotoxicity of AraC (Fig. 1B). Yet, dC itself did not promote the survival of MTNs grown without any trophic support (data not shown). The potency of dC inhibition of AraC neurotoxicity was next determined by treating established cultures with AraC (1 mM) for 5 days in the presence of various concentrations of dC. MTNs treated with ,100 nM dC were dead, looking similar to those treated with AraC alone. Cultures treated with AraC and concentrations of dC ranging from 1 to 100 mM were alive and looked healthy. At concentrations .1 mM, dC was clearly toxic. The EC50 of dC for preventing the neurotoxicity of 1 mM AraC on chick embryo spinal cord MTNs was about 100 nM (data not shown). AraC and dC, as well as other nucleosides and deoxynucleosides, gain access to the
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inside of cells by facilitated diffusion through a well characterised membrane transporter [16]. Such transport can be prevented by the specific inhibitor nitrobenzylthioinosine (NBTI) [16]. Interestingly, NBTI has been shown to block the neurotoxic effect of AraC in ciliary and dorsal root ganglion neurons [15]. Similarly, we also found that NBTI (10 mM) was able to block the neurotoxic effect of AraC (1 mM) on cultured MTNs (Fig. 1B). Moreover, NBTI by itself was not toxic to MTNs grown in the presence of MEX (data not shown). These results suggest that AraC enters the neuron via a NBTI-sensitive membrane transporter. We have previously shown that spinal cord MTNs depend for survival on trophic activities present in muscle tissue, their target of innervation [2]. In this study, we found strong similarities between the MTN cell death induced by AraC in vitro and that following MEX deprivation, in agreement with what has been reported for other neuronal types dependent for survival on exogenous trophic factors [3,8,12,15]. All this evidence favours the view that a similar ‘death program’ could become activated in both paradigms. Interestingly, several proteins (including heat-shock proteins, cytoskeleton-associated proteins, and others of unknown identity) are oversynthesized in AraC-treated neurons [3], some of which could be similar to those described in trophic factor-deprived neurons [13]. Yet, AraC may also have independent effects from neurotrophic factor deprivation. For instance, it has been hypothesised that the observed delay in onset of neuronal death triggered by AraC as compared to neurotrophic deprivation (Fig. 2), could be due to the specific targeting by AraC of an intermediate step [8]. Moreover, the fact that the neurotoxicity of AraC can be blocked specifically by its analogue dC (Fig. 1) [3,8,12,15], suggests that AraC may interfere with a dC-dependent process necessary for neuronal survival. Since AraC is known to interfere with intracellular phosphorylation of deoxynucleosides, it has been hypothesised that AraC may alter the intracellular levels of dCTP in a specific manner [8,15], which in turn may be critical for the survival of postmitotic neurons. AraC could also act by interfering with the DNA repair system [4,12], thus causing apoptosis. In summary, our results reinforce the relevance of purine and pyrimidine metabolism to the survival of neuronal systems. In fact, the existence of a biochemical mechanism for the induction of neuronal death by an endogenous deoxynucleoside in the exogenous trophic factor support, has recently been documented [14]. Further studying of these metabolic pathways will surely provide important insights into mechanisms of neuronal survival relevant to the development of the nervous system as well as in pathologic neurodegeneration.
The Unit of Molecular Neurobiology is currently supported by grants of FIS (94/1576), Cancer Telemarato´ de TV3, Ajuntament de Lleida and Biomed2 and Biotech programs of the European Union. [1] Bustos, M., Alcain, F.J. and Padilla, F., Survival of chick embryo ciliary ganglia neurons in cellular cultures. Collagen, polylysine, and cytosine-arabinoside effects, Trab. Inst. Cajal, 71 (1980) 63– 70. [2] Comella, J.X., Sanz-Rodriguez, C., Aldea, M. and Esquerda, J.E., Skeletal muscle-derived trophic factors prevent motoneurons from entering an active cell death program in vitro, J. Neurosci., 14 (1994) 2674–2686. [3] Dessi, F., Pollard, H., Moreau, J., Ben-Ari, Y. and CharriautMarlangue, C., Cytosine arabinoside induces apoptosis in cerebellar neurons in culture, J. Neurochem., 64 (1995) 1980–1987. [4] Fram, R.J. and Kufe, D.W., Inhibition of DNA excision repair and the repair of x-ray-induced DNA damage by cytosine arabinoside and hydroxyurea, Pharmacol. Ther., 31 (1985) 165–176. [5] Gorczyca, W., Bigman, K., Mittelman, A., Ahmed, T., Gong, J., Melamed, M. and Darzynkiewicz, Z., Induction of DNA strand breaks associated with apoptosis during treatment of leukemias, Leukemia, 7 (1993) 659–670. [6] Hwang, T.L., Yung, W.K.A., Estey, E.H. and Fields, W.S., Central nervous system toxicity with high doses Ara-C, Neurology, 35 (1985) 1475–1479. [7] Kufe, D.W. and Major, P.P., Studies on the mechanism of action of cytosine arabinoside, Med. Pediatr. Oncol., 1 (1982) 49–67. [8] Martin, D.P., Wallace, T.L. and Johnson, E.M. Jr., Cytosine arabinoside kills postmitotic neurons in a fashion resembling trophic factor deprivation: evidence that a deoxycytidine-dependent process may be required for nerve growth factor signal transduction, J. Neurosci., 10 (1990) 184–193. [9] Masuck, T.M., Taylor, A.R. and Lough, J., Arabinosylcytosineinduced accumulation of DNA nicks in myotube nuclei detected by in situ nick translation, J. Cell Physiol., 144 (1990) 12– 17. [10] Russell, J.A. and Powles, R.L., Neuropathy due to cytosine arabinoside, Br. Med. J., 4 (1974) 652–653. [11] Smith, R.A. and Orr, D.J., The survival of adult mouse sensory neurons in vitro is enhanced by natural and synthetic substrata, particularly fibronectin, J. Neurosci., 17 (1987) 265–270. [12] Tomkins, C.E., Edwards, S.N. and Tolkovsky, A.M., Apoptosis is induced in post-mitotic rat sympathetic neurons by arabinosides and topoisomerase II inhibitors in the presence of NGF, J. Cell Sci., 107 (1994) 1499–1507. [13] Villa, P., Miehe, M., Sensenbrenner, M. and Pettmann, B., Synthesis of specific proteins in trophic-deprived neurons undergoing apoptosis, J. Neurochem., 62 (1994) 1468–1475. [14] Wakade, A.R., Przywara, D.A., Palmer, K.C., Kulkarni, J.S. and Wakade, T.D., Deoxynucleoside induces neuronal apoptosis independent of neurotrophic factors, J. Biol. Chem., 270 (1995) 17986– 17992. [15] Wallace, T.L. and Johnson, E.M. Jr., Cytosine arabinoside kills postmitotic neurons: evidence that deoxycytidine may have a role in neuronal survival that is independent of DNA synthesis, J. Neurosci., 9 (1989) 115–124. [16] Young, J.D. and Jarvis, S.M., Nucleoside transport in animal cells, Biosci. Rep., 3 (1983) 309–322.
Cytosine arabinoside is neurotoxic to chick embryo spinal cord motoneurons in culture Cesar Sanz-Rodriguez1, Jacint Boix, Joan X. Comella* Unitat de Neurobiologia Molecular, Departament de Cie`ncies Me`diques Ba`siques, Facultat de Medicina, Universitat de Lleida, Rovira Roure 44, 25198 Lleida, Spain Received 23 December 1996; revised version received 22 January 1997; accepted 22 January 1997
Abstract Cytosine arabinoside (1-b-d-arabinofuranosylcytosine, AraC) is a commonly used antimitotic agent that kills proliferating cells by inhibiting DNA synthesis. We report that AraC is toxic to cultured chick embryo spinal cord motoneurons (MTNs) in a concentrationdependent fashion with an EC50 of about 2 mM. Interestingly, this type of MTN death is specific, resembles that occurring upon muscle extract (MEX) trophic deprivation regarding its morphological and temporal characteristics, and has apoptotic features, as judged by observation of nuclear morphology. The death of AraC-treated MTNs can be blocked by 2′-deoxycytidine (dC), a pyrimidine metabolite AraC is structurally related to. Overall, these findings suggest that dC may participate in a pathway, different from inhibition of DNA synthesis, that is necessary for cultured MTNs to respond to the trophic activities present in MEX. 1997 Elsevier Science Ireland Ltd. Keywords: Motoneurons; Muscle extract; Cytosine arabinoside; Cell death; Apoptosis; Nitrobenzylthioinosine; 2′-Deoxycytidine
Cytosine arabinoside (1-b-d-arabinofuranosylcytosine, AraC) is a pyrimidine antimetabolite structurally related to 2′-deoxycytidine (dC). It is a useful tool to selectively kill dividing cells, since it may be incorporated into DNA, thus causing inhibition of DNA synthesis [7]. Indeed, AraC is widely used in tissue culture of non-mitotic cells to eliminate proliferating cells, such as fibroblasts or glia, as well as a chemotherapeutic agent for certain lymphoproliferative disorders. Yet, AraC may also affect certain non-dividing cells. In vitro, AraC is more toxic to neurons than other antimitotic drugs [1,11]. Moreover, cancer patients treated with AraC can present with central and peripheral nervous system degeneration [6,10]. Several researchers have used in vitro models to examine the mechanisms underlying the neurotoxicity of AraC. This drug blocks specifically the survival in vitro of postmitotic parasympathetic, sympathetic, and sensory neurons stimu* Corresponding author. Tel.: +34 73 702438; fax: +34 73 702426; e-mail: [email protected] 1 Present address: Servicio de Hematologı´a, Hospital Universitario de la Princesa, 28006 Madrid, Spain.
lated by neurotrophic factors [8,12,15] as well as of cerebellar neurons [3]. The neuronal death induced by AraC is apoptotic [3,8,12], in agreement with what has been reported for other cell types [5,9]. AraC appears to exert its neurotoxic effect by interfering with a dC-dependent step included in neurotrophic factor signalling pathways, which is independent of DNA synthesis or repair [8,15]. In this study, we investigated whether AraC is also neurotoxic to postmitotic spinal cord motoneurons (MTNs). MTNs were purified from 5.5-day-old chick embryos (COPAGA, Spain) as previously reported [2]. First, spinal cord MTNs that had been cultured for 24 h in the presence of muscle extract (MEX), were treated with varying concentrations (10 nM–100 mM) of AraC while in the presence of MEX for 5 additional days. Both MTN survival and morphological changes occurring during the experiment were assessed in detail. At very high concentrations of AraC (100 mM), MTNs degenerated very rapidly while swelling and accumulating vacuoles, in what clearly looked like a dose-related and non-specific toxic effect. However, at concentrations of AraC ranging from 10 nM to 10 mM, no changes were observed in the exposed cul-
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Fig. 1. The neurotoxic effect of AraC on spinal cord MTNs is potent (EC50 ca. 2 mM) (A) and specific, and can be prevented by dC and NBTI (B). MTNs were seeded at a density of 10 000/well in 96-well culture dishes, and grown for 24 h in the presence of MEX (300 mg/ml). At that time, cells in the central area of every culture well were counted at 20 × magnification with phase-contrast inverted microscopy. Cells considered to be alive, bore neurites longer than 2 cell diameters. For each experiment, the value taken as the corrected 100% survival, represented the average of 6–8 wells. Thereafter, culture wells were washed and next exposed to fresh medium containing appropriate concentrations of the drugs. In a first series of experiments, the potency of AraC neurotoxicity was determined by exposing MTN cultures to concentrations of AraC ranging from 10 nM to 100 mM (A). In a second series of experiments (B), MTNs grown in the presence of MEX were treated with dC (1 mM) or NBTI (10 mM) in the presence of AraC (1 mM), aphidicolin (1 and 10 mM), or 5′-fluorodeoxyuridine (1 and 10 mM). Additional groups of MTNs were always maintained in MEX alone or without any trophic support (Depr). MTN survival was evaluated in the same microscopic field 5 days later. Survival was expressed as a percentage of neuronal counts with respect to the previously established 100% value. Each point represents the mean ± SEM of 6–8 wells from a single experiment that was repeated twice more with results comparable to those presented (*P , 0.05, significantly different from control, MEX-treated cultures using the Mann–Whitney U-Wilcoxon test). The different supplements and drugs used were obtained from Sigma. Tissue culture dishes and culture media were obtained from Corning and Gibco, respectively.
tures over the first 48 h. By 72 h, MTNs exposed to .1 mM AraC began to show signs of atrophy, which was followed by dramatic degeneration and condensation of the cell bodies over the following 24–48 h. The neurites became progressively thinner and discontinuous. After 5 days of AraC treatment, the culture dish was covered with degenerated, phase-dark residua of cell bodies and neurites. At concentrations of AraC ,0.5 mM, MTNs looked healthy. When incubated in concentrations of AraC between 0.5 and 1 mM, MTNs appeared somewhat atrophic and partially degenerated, even though many of them were still alive. Quantitative analysis of survival revealed an EC50 of approximately 2 mM, indicating that spinal cord MTNs were very sensitive to AraC (Fig. 1A). Cultured MTNs were also incubated in the presence of two other antimitotic drugs, aphidicolin and 5′-fluorodeoxyuridine, whose ability at killing proliferating cells is comparable to that of AraC. These agents inhibit DNA synthesis and repair, respectively, thus sharing with AraC the same general mechanism of action. None of these drugs (which were both used at 1 and 10 mM for 5 days) had any significant
neurotoxic effect on MTNs (Fig. 1B), suggesting that the cytotoxic effect of AraC was specific and not just the result of a general toxic effect since similar concentrations of AraC were clearly neurotoxic. Under phase-contrast microscopy, the morphology of AraC-treated cultured MTNs closely resembled that of MTNs deprived of MEX [2]. To compare these deaths more carefully, we compared the time courses of death caused by MEX deprivation and by AraC (1 mM) treatment (Fig. 2). Interestingly, the kinetics of death induced by AraC were similar to MEX deprivation, though delayed in onset about 48 h. During that delay period, MTNs remained apparently unaffected. In both cases, the extent of MTN death correlated with the morphological degeneration of the cells. We have previously demonstrated that the death of cultured chick embryo spinal cord MTNs upon MEX deprivation is apoptotic [2]. The close resemblance found between MTN death induced by AraC and that caused by trophic deprivation, prompted us to examine the nuclear morphology of dying AraC-treated cultures. MTNs that had been grown for 48 h in the presence of MEX, were exposed to AraC (1 mM) for varying lengths of time (from 24 to 120 h after addition of AraC). Control cultures either in the presence or in the absence of MEX were also maintained. MTNs were next fixed, stained with the apoptotic-specific Hoescht 33258 dye, and finally observed with a microscope equipped with fluorescence and UV filters. Parallel examination of the same microscopic field with Nomarsky optics allowed the precise identification and, thus, quantitation of those neurons
Fig. 2. Both MEX deprivation (filled triangles) and AraC (filled squares) cause the rapid degeneration of MTNs over a 24-h period following an initial lag in which neurons appear unaffected. MEX-treated cultures (open circles) showed a very high survival response over the culture time. The method to evaluate cell survival was the same as in Fig. 1. At times indicated on the abscissa, survival was evaluated. Survival was expressed as the percentage of neurons remaining with respect to the number of neurons present in the culture when drugs were added. Each point represents the mean ± SEM of 6–8 wells from a single experiment that was repeated twice with results comparable to those presented.
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Fig. 3. AraC-induced death of MTNs is apoptotic: (A) quantitative and (B) morphological evaluation. MTNs were cultured at a density of 150 000 cells in 35 mm culture plates. After MTNs had grown for 48 h in the presence of MEX (300 mg/ml), cultures were exposed to AraC (1 mM) (filled circles). An additional group of MTNs was always maintained in the presence of MEX (open squares), while a third group was deprived of trophic support (open circles). At 24, 48, 72, 96, and 120 h after addition of AraC, MTNs were fixed at 4°C with 2.5% (v/v) glutaraldehyde in phosphate-buffered saline (pH 7.4) for 30 min. MEX-treated cultures were processed 5 days after the establishment of the conditions and deprived cultures after 24 h. Thereafter, MTNs were stained for 1 h with 0.05 mg/ml Hoescht 33258 dye. The culture plates containing the neurons were coverslipped and counted with a vertical microscope equipped with epifluorescence and UV filters. Parallel examination of the same microscopic field with Nomarsky optics allowed the precise identification of apoptotic neurons. (A) Apoptotic MTNs were counted at 20 × magnification. The cell counts for each experimental condition consisted of 6 fields/slide and 3 slides/ condition. Values represented are the mean ± SEM of three replicates from a single experiment that was repeated twice more with results comparable to those presented. MEX-treated cultures of MTNs untreated (a) and treated (b) with AraC were viewed with epifluorescence optics after Hoescht 33258 staining. A clear increase in the number of MTNs undergoing apoptosis in response to the AraC treatment could easily be appreciated. Arrows indicate apoptotic neurons, whose DNA has condensed into brightly stained masses. Scale bar, 40 mm.
that exhibited an apoptotic morphology, i.e. those cells exhibiting micronucleation of their DNA and those that had become ghost cells due to the entire degradation of their DNA. After 4–5 days in AraC-treated cultures 10– 15% of MTNs displayed apoptotic nuclear morphologies (Fig. 3A). The nuclei appeared condensed and with a brighter fluorescence than normal nuclei, indicating that chromatin had aggregated and collapsed at the nuclear membrane. Condensation of the chromatin at the nuclear membrane was followed by fragmentation of the nucleus (Fig. 3B). It should be noted that 3–4% of MTNs were found to apoptose spontaneously in cultures exposed to MEX only, while this value increased to about 9% in MEX-deprived cultures (Fig. 3A). These results suggested that AraC-induced MTN death was most likely apoptotic. AraC neurotoxicity seems to involve a process specific for dC. This hypothesis was based upon the observation that dC specifically counters the neurotoxic effect of AraC
in several neuronal types [8,12,15]. In order to validate these results for embryonic MTNs, established cultures were treated with AraC (1 mM) in the presence of an equimolar concentration of dC. After 5 days, dC entirely blocked the neurotoxicity of AraC (Fig. 1B). Yet, dC itself did not promote the survival of MTNs grown without any trophic support (data not shown). The potency of dC inhibition of AraC neurotoxicity was next determined by treating established cultures with AraC (1 mM) for 5 days in the presence of various concentrations of dC. MTNs treated with ,100 nM dC were dead, looking similar to those treated with AraC alone. Cultures treated with AraC and concentrations of dC ranging from 1 to 100 mM were alive and looked healthy. At concentrations .1 mM, dC was clearly toxic. The EC50 of dC for preventing the neurotoxicity of 1 mM AraC on chick embryo spinal cord MTNs was about 100 nM (data not shown). AraC and dC, as well as other nucleosides and deoxynucleosides, gain access to the
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inside of cells by facilitated diffusion through a well characterised membrane transporter [16]. Such transport can be prevented by the specific inhibitor nitrobenzylthioinosine (NBTI) [16]. Interestingly, NBTI has been shown to block the neurotoxic effect of AraC in ciliary and dorsal root ganglion neurons [15]. Similarly, we also found that NBTI (10 mM) was able to block the neurotoxic effect of AraC (1 mM) on cultured MTNs (Fig. 1B). Moreover, NBTI by itself was not toxic to MTNs grown in the presence of MEX (data not shown). These results suggest that AraC enters the neuron via a NBTI-sensitive membrane transporter. We have previously shown that spinal cord MTNs depend for survival on trophic activities present in muscle tissue, their target of innervation [2]. In this study, we found strong similarities between the MTN cell death induced by AraC in vitro and that following MEX deprivation, in agreement with what has been reported for other neuronal types dependent for survival on exogenous trophic factors [3,8,12,15]. All this evidence favours the view that a similar ‘death program’ could become activated in both paradigms. Interestingly, several proteins (including heat-shock proteins, cytoskeleton-associated proteins, and others of unknown identity) are oversynthesized in AraC-treated neurons [3], some of which could be similar to those described in trophic factor-deprived neurons [13]. Yet, AraC may also have independent effects from neurotrophic factor deprivation. For instance, it has been hypothesised that the observed delay in onset of neuronal death triggered by AraC as compared to neurotrophic deprivation (Fig. 2), could be due to the specific targeting by AraC of an intermediate step [8]. Moreover, the fact that the neurotoxicity of AraC can be blocked specifically by its analogue dC (Fig. 1) [3,8,12,15], suggests that AraC may interfere with a dC-dependent process necessary for neuronal survival. Since AraC is known to interfere with intracellular phosphorylation of deoxynucleosides, it has been hypothesised that AraC may alter the intracellular levels of dCTP in a specific manner [8,15], which in turn may be critical for the survival of postmitotic neurons. AraC could also act by interfering with the DNA repair system [4,12], thus causing apoptosis. In summary, our results reinforce the relevance of purine and pyrimidine metabolism to the survival of neuronal systems. In fact, the existence of a biochemical mechanism for the induction of neuronal death by an endogenous deoxynucleoside in the exogenous trophic factor support, has recently been documented [14]. Further studying of these metabolic pathways will surely provide important insights into mechanisms of neuronal survival relevant to the development of the nervous system as well as in pathologic neurodegeneration.
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