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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 H ,7 H -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.

Neurons in the developing brain die via apoptosis after DNA damage, while neurons in the adult brain are generally resistant to these insults. The basis for this resistance is a matter of conjecture. We report here that cerebellar granule neurons (CGNs) in culture lose their competence to die in response to DNA damage as a function of time in culture. CGNs at either 1 day in vitro (DIV) or 7 DIV were treated with the DNA damaging agents camptothecin, UV or c-irradiation and neuronal survival measured. The younger neurons were effectively killed by these agents, while the older neurons displayed a significant resistance to killing. Neuronal survival did not change with time in culture when cells were treated with C2-ceramide or staurosporine, agents which do not target DNA. The resistance to UV irradiation developed over time in culture and was not due to changes in mitotic rate. Increases in DNA strand breakage, up-regulation of the levels of both p53 and its phosphorylated form and nuclear translocation of p53 were equivalent in both older and younger neurons, indicating a comparable p53 stress response. In addition, we show that treatment of older neurons with pharmacological inhibitors of distinct components of the DNA repair machinery promotes the accumulation of DNA damage and sensitizes these cells to the toxic effects of UV exposure. These data demonstrate that older neurons appear to be more proficient in DNA repair in comparison to their younger counterparts, and that this leads to increased survival after DNA damage.

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