The Journal of Neuroscience, January 1989, 9(l): 115-124 Cytosine Arabinoside Kills Postmitotic Neurons: Evidence That Deoxycytidine May Have a Role in Neuronal Survival That Is Independent of DNA Synthesis Thomas L. Wallace’ and Eugene M. Johnson, Jr.2 ‘Center for Biotechnology, Baylor College of Medicine, and Houston Biotechnology Incorporated, The Woodlands, Texas 77381, and ‘Department of Pharmacology, Washington University School of Medicine, St. Louis, Missouri 63110 Cytosine arabinoside (ARA C), a competitive inhibitor of the independent of DNA synthesis or repair. Thus, 2’-deoxycy- incorporation of P’-deoxycytidine into DNA in other cell types, tidine may have an important and previously unrecognized caused a concentration-dependent inhibition of KCI- and in- role in cellular function that in the case of neurons is critical sulin-stimulated survival of postmitotic ciliary parasympa- for survival. thetic ganglion neurons, and the nerve growth factor (NGF)- stimulated survival of postmitotic dorsal root ganglion (DRG) Neuronal survival dependsupon extracellular neurotrophic fac- sensory neurons in vitro. The IC,, for survival was 2 x 1O-8 tors derived from target tissues,and many such factors are be- M for both types of neurons after 4 d under the culture con- lieved to exist, each directed toward a specific neuronal type or ditions used. The inhibition of DRG survival by ARA C in the several types. Nerve growth factor (NGF) has been shown to presence of varying concentrations of NGF indicated that be important in vivo for the differentiation and survival of sym- ARA C acted as an apparent noncompetitive antagonist of pathetic, dorsal root ganglion (DRG) sensory,and probably cer- NGF. This cytotoxic effect of ARA C was blocked by 2’- tain CNS cholinergic neurons (Levi-Montalcini and Angeletti, deoxycytidine, but not by cytosine, 2’-deoxyadenosine, 2’- 1968; Johnson et al., 1980; Hefti et al., 1985). A number of deoxyguanosine, or 2’-deoxythymine, indicating that ARA C other factors have been highly purified and shown to have sur- was interfering with a deoxycytidine-specific survival pro- vival-promoting effectson specificneuronal types in vitro (Barde cess. Cytidine could block ARA C toxicity, but it was 40 times et al., 1982; Barbin et al., 1984; Morrison et al., 1986; Wallace less potent than 2’-deoxycytidine. The blockade of the cy- and Johnson, 1987). The molecular mechanismby which these totoxic effect of ARA C by L’-deoxycytidine indicated that factors, even the well-studied NGF, maintain the survival of P’-deoxycytidine was an apparent competitive antagonist of neurons is not understood, but it is possiblethat a number of ARA C toxicity. 2’-Deoxycytidine, by itself, was not survival- different neurotrophic factors share common intracellular me- promoting. Other antimitotic agents, such as adenine ara- diators. A number of substanceshave been identified that may binoside, thymine arabinoside, Sfluorodeoxyuridine, S-bro- have a role in neuronal survival and include the Na+,K+ pump modeoxycytidine, 5-azadeoxycytidine, and aphidicolin had (Skaper et al., 1986) protein kinase C (Montz et al., 1985) no effect on neuronal survival at a concentration 5000 times methyltransferase (Acheson et al., 1986) CAMP (Rydel and the EC,, of ARA C, indicating that inhibition of DNA synthesis Greene, 1988) and ras p21 (Hagag et al., 1986). or repair was probably not the mechanism by which ARA C We report in this paper data suggestingthat 2’-deoxycytidine, inhibited neuronal survival and that other P’-deoxynucleo- in contrast to other 2’-deoxynucleosides,may be a critical factor sides were not involved in the survival-promoting process. in the survival of postmitotic peripheral neurons and that this Nitrobenzylthioinosine, an inhibitor of 2’-deoxycytidine and involvement appearsto be independent of DNA synthesisor ARA C membrane transport in other cell types, inhibited the repair. Cytosine arabinoside (ARA C), which is a structural cytotoxic effect of ARA C in neurons, suggesting that ARA analogof 2’-deoxycytidine, had potent cytotoxic effectson para- C entered the neurons through a similar transport mecha- sympathetic and DRG sensory neurons, whereas other anti- nism and that ARA C needed to gain access to the inside metabolite DNA synthesisinhibitors had no effect on neuronal of the neuron to be effective. These results indicate that survival at SOOO-foldhigher concentrations.ARA C differs from ARA C, in addition to being an antimitotic agent for dividing 2’-deoxycytidine by having an arabinosesugar in place of the cells, is also cytotoxic for postmitotic neurons. This inhibition 2’-deoxyribose sugar.This indicates that 2’-deoxycytidine is im- of neuronal survival by ARA C is hypothesized to be due to portant in neuronal survival and that its role in cellular function inhibition of a 2’-deoxycytidine-dependent process that is may extend beyond that of a metabolic precursor of DNA. Materials and Methods Received Nov. 17, 1987; revised June 7, 1988; accepted June 8, 1988. We wish to thank Ms. Patricia Osborne for her technical assistance. This work Materials. Insulin was a gift of Eli Lilly Co. (Indianapolis, IN). Eagle’s was supported by PHS Research Grants NS18071 and PSO-AG05681. T.L.W. minimumessential medium (MEM), fetal calf serum, and trypsin were was supported, in part, by PHS Training Grant T32NS07 129. purchased from Gibco (Grand Island, NY). Collagenase (type 4) was Correspondence should be addressed to Thomas L. Wallace, Ph.D., Baylor purchased from Cooper Biomedical (Malvem, PA). NGF (2%) was College of Medicine, Center for Biotechnology, 4000 Research Forest Drive, The prepared by the method of Bocchini and Angeletti (1969). All other Woodlands, TX 7738 1. reagents were purchased from Sigma Chemical Co. (St. Louis). Copyright 0 1989 Society for Neuroscience 0270-6474/89/010115-10$02.00/O Methods. Parasympathetic ciliary or DRG were taken from chicken 116 Wallace and Johnson . Deoxycytidine-Dependent Neuronal Survival embryos that had been incubated for 8 d (E8) at 37°C. This age is one Berg (198 1). In the presenceof 35 mM KCl, insulin causeda at whichthe neuronsare postmitotic.The birthdate(time at whichthe logarithmic increasein survival up to 500 mU insulin/ml, rep- neuronundergoes terminal mitosis) is E4.5 (ciliarv) and E5 (DRG) in chickens(Rohrer and Thoenen,1987). E8 neurons.I were used ~ because I resenting a 3.5-fold increase at the highest concentration. By very fewnon-neuronal cells were obtained upon plating at this age,even contrast, a 25,000 x g supernatant fraction of pig lung caused in the absenceof antimitoticagents. Ciliary gangliawere digested by a an 8-fold increasein neuronal survival in the sameexperiment. 30 minincubation at 37°Cin trypsin (0.0125%), and DRG weredigested We have previously reported pig lung to contain high concen- by a 20 min incubationat 37°Cwith collagenase(type 4) followedby trations of parasympathetic neurotrophic activity (Wallace and a 10 min incubationat 37°Cwith trvnsin (0.0625%).Ganglia were dissociatedinto singlecells by trituration with‘a small-borepiiet. Neu- Johnson, 1986, 1987). With insulin, maximal survival was ap- ronswere incubated at 37°Cin 0.35 ml of mediumon a doublelayer parently not achieved, even at the highest concentration of in- of air-dried,rat tail collagenin 48 well plasticdishes (Costar 3548) in sulin used(500 mu/ml; 6.5 x 1O-6M). Insulin causedthe out- 95%air, 5% CO>.The mediumincluded Eagle’s MEM, 100units/ml growth of neurites from someciliary neurons, but this was not of penicillin, 100 &ml of streptomycin,and 10%fetal calf serum. Insulin servedas the trophic factor for ciliary neuronsand NGF for characterized further. Insulin at a concentration of 50 mu/ml, DRG neurons.KC1 (35 mM) wasalso included in the ciliary neuron which gives a 3-fold stimulation of survival, was used as the medium.Plating efficiency, determined 2 hr after addingthe neurons trophic factor to maintain survival of neurons in further ex- to the wells,was usually about 70%. periments using ciliary neurons. The actual importance of in- Ciliarv and DRG neuronswere culturedfor 4 d at 37°Cand then sulin as an in vivo neurotrophic factor for parasympatheticneu- evaluatedfor survival.Neurons were fixed in 2%glutaraldehyde in PBS, andthe meannumber of survivingneurons was determined in 15fields rons is questionablebecause of its relatively low concentration usingan inverted microscopewith a phase-contrastobjective (Barbin in the blood compared with the concentration needed to see et al., 1984).Neurons could easily be distinguishedfrom non-neurons significant neuronal survival. It was simply usedhere as a pur- in thesecultures. Viable neurons were round and phasebright, whereas ified survival-promoting factor for further studies on the role deadneurons were crenated and phasedark. Survival wasexpressed as the meannumber of surviving neuronsper well. of 2’-deoxycytidine in the survival of ciliary neurons. Concentration-responseeffect of ARA C on the survival of Results parasympathetic neurons Efect of insulin on the survival of parasympathetic neurons As previously discussed,2’-deoxycytidine was one of the com- The rationale for the experiments presented in this paper is ponents of the HBlO 1 medium that had survival-promoting basedon a serendipitousinitial finding. Experiments were being effects on ciliary neurons under the culture conditions used, performed in which various cell types were being grown in a which included KC1 (35 mM) and ARA C (10m5M). Normally, defined, serum-free medium (HB 10 1; Hana Biologicals, Inc.). it would be predicted that ARA C (and other agentsthat inhibit The conditioned medium from thesecells was being tested for DNA synthesis)would not exert deleteriouseffects on postmi- survival-promoting activity on ciliary neurons incubated in a totic neurons. An experiment investigating the concentration- medium that would not support neuronal survival. The ciliary responseeffect of ARA C on survival of ciliary neurons was neuron medium contained Eagle’sMEM, 10% serum, antibiot- performed in which E8 chicken ciliary neurons were incubated its, KC1 (35 mM), and ARA C (1 O-5M).