Cytosine arabinoside kills postmitotic neurons: evidence that deoxycytidine may have a role in neuronal survival that is independent of DNA synthesis

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Abstract
Cytosine arabinoside (ARA C), a competitive inhibitor of the incorporation of 2′-deoxycytidine into DNA in other cell types, caused a concentration-dependent inhibition of KCl- and insulin-stimulated survival of postmitotic ciliary parasympathetic ganglion neurons, and the nerve growth factor (NGF)-stimulated survival of postmitotic dorsal root ganglion (DRG) sensory neurons in vitro. The IC50 for survival was 2 x 10(-8) M for both types of neurons after 4 d under the culture conditions used. The inhibition of DRG survival by ARA C in the presence of varying concentrations of NGF indicated that ARA C acted as an apparent noncompetitive antagonist of NGF. This cytotoxic effect of ARA C was blocked by 2′-deoxycytidine, but not by cytosine, 2′- deoxyadenosine, 2′-deoxyguanosine, or 2′-deoxythymine, indicating that ARA C was interfering with a deoxycytidine-specific survival process. Cytidine could block ARA C toxicity, but it was 40 times less potent than 2′-deoxycytidine. The blockade of the cytotoxic effect of ARA C by 2′-deoxycytidine indicated that 2′-deoxycytidine was an apparent competitive antagonist of ARA C toxicity. 2′-Deoxycytidine, by itself, was not survival-promoting. Other antimitotic agents, such as adenine arabinoside, thymine arabinoside, 5-fluorodeoxyuridine, 5- bromodeoxycytidine, 5-azadeoxycytidine, and aphidicolin had no effect on neuronal survival at a concentration 5000 times the EC50 of ARA C, indicating that inhibition of DNA synthesis or repair was probably not the mechanism by which ARA C inhibited neuronal survival and that other 2′-deoxynucleosides were not involved in the survival-promoting process. Nitrobenzylthioinosine, an inhibitor of 2′-deoxycytidine and ARA C membrane transport in other cell types, inhibited the cytotoxic effect of ARA C in neurons, suggesting that ARA C entered the neurons through a similar transport mechanism and that ARA C needed to gain access to the inside of the neuron to be effective. These results indicate that ARA C, in addition to being an antimitotic agent for dividing cells, is also cytotoxic for postmitotic neurons. This inhibition of neuronal survival by ARA C is hypothesized to be due to inhibition of a 2′-deoxycytidine-dependent process that is independent of DNA synthesis or repair. Thus, 2′-deoxycytidine may have an important and previously unrecognized role in cellular function that in the case of neurons is critical for survival.