EXPERIMENTAL

CELL RESEARCH

197,

75-81

(1991)

Deoxyadenosine- and Cyclic AMP-Induced Cell Cycle Arrest and Cytotoxicity D.A.ALBERT,‘E. Department

of Medicine,

NODZENSKI,G.HEREDIACRUZ, J. KUCHIBHOLTLA,AND J. KOWALSKI

University

of Chicago Medical

Center, 5841 South Maryland

INTRODUCTION The Gl phase of the cell cycle is a period of varying length during which a complex series of events that prepare the cell for DNA synthesis occur. These include ion fluxes, transcriptional activation of immediate early genes, and subsequent activation of genes whose products participate in intermediary metabolism [l]. These phenomena appear to be necessary for progress through the cell cycle since their inhibition can generate Gl cell cycle arrest. In addition, a number of agents with less clearly defined mechanisms of action including direquests should he addressed.

60637

methyl sulfoxide [2] and mimosine [3], a plant amino acid, result in Gl cell cycle arrest. Two other agents with partially defined mechanisms of action that result in Gl arrest are deoxyadenosine [4] and cyclic AMP [5]. Cyclic AMP-induced Gl arrest of S49 cells, as is true of all the known effects of cyclic AMP in eukaryotic cells, is mediated by cyclic AMP-dependent protein kinase (PKA) [6]. PKA phosphorylates serine and threonine residues on a variety of proteins including its own regulatory subunit [ 71,the M2 subunit of ribonucleotide reductase [8], and a recently described 43-kDa protein, the cyclic AMP response element binding protein (CREB) [9, lo]. This protein is thought to transduce transcriptional activation generated by cyclic AMP. How phosphorylation results in Gl phase cell cycle arrest in T lymphocytes is unknown at this time. Deoxyadenosine also causes Gl cell cycle arrest [ll] and has a number of putative toxic effects on lymphocytes, including (i) inhibition of pyrimidine biosynthesis [I2], (ii) inhibition of S-adenosylmethionine-dependent methylation reactions [ 131, (iii) ATP depletion [ 14-161, (iv) NAD+ depletion [17], (v) deoxyadenosine triphosphate-induced inhibition of ribonucleotide reductase [18], and other effects. Which of these mechanisms is responsible for Gl cell cycle arrest is also presently unknown. One study has suggested that deoxyadenosine is toxic to cells because it increases cyclic AMP concentrations [19]. We obtained deoxyadenosine-resistant cell lines by exposing wild type S49 T lymphoma cells to progressively higher concentrations of hydroxyurea. Hydroxyurea is an inhibitor of ribonucleotide reductase that binds to the tyrosyl radicals on the M2 subunit of the enzyme [20], and exposure of cells to hydroxyurea generates hydroxyurea-resistant cell lines that have increased ribonucleotide reductase activity due, in part, to amplification of the genes that code for the enzyme. These cell lines are resistant to the cytotoxicity of deoxyadenosine proportional to the degree of elevation of their deoxynucleoside triphosphate pools [4]. This suggests that deoxyadenosine cytotoxicity might be mediated by dATP inhibition of ribonucleotide reductase with resultant depletion of other deoxynucleoside triphosphate pools. Because these cell lines are also par-

We compared deoxyadenosine (AdR)- and cyclic AMP (CAMP)-induced cell cycle arrest and cytotoxicity in wild type and mutant 549 cells to determine whether they resulted from the same or different mechanisms. Cyclic AMP and deoxyadenosine are synergistic rather than additive in cytotoxicity assays, suggesting different mechanisms of toxicity. Although cyclic AMP causes cell death after 72 h, in concentrations sufficient to result in cell cycle arrest it is reversible with virtually no cytotoxicity for at least 24 h, whereas AdR-induced cell cycle arrest is lethal and irreversible. AdRinduced Gl cell cycle arrest results in diminished ribonucleotide reductase activity but the kinetics of this inhibition differ from cyclic AMP-induced cell cycle arrest. Cyclic AMP arrest and cytotoxicity depend on cyclic AMP-dependent protein kinase (PKA) activity, whereas AdR toxicity does not differ between cell lines with or without PKA activity. Furthermore, deoxycytidine prevents AdR cell cycle arrest and cytotoxicity but has no effect on cyclic AMP Gl arrest. Finally, comparison of cytofluorographic patterns of Gl-arrested cells suggests that the AdR block is later in Gl than cyclic AMP-induced cell cycle arrest. In summary, these data show that while the mechanisms of cell cycle arrest and cytotoxicity of cyclic AMP and deoxyadenosine are uncertain, they do appear to involve different pathways. Q 1991 Academic Press, Inc.

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76

ALBERT

tially resistant to Gl arrest by deoxyadenosine (A&) we hypothesized that AdR-induced Gl cell cycle arrest may also be mediated by ribonucleotide reductase. Against this explanation are the observations that ribonucleotide reductase activity is virtually absent in Gl phase cells [21] and that other inhibitors of ribonucleotide reductase, such as hydroxyurea, result in S phase inhibition. Since cyclic AMP and deoxyadenosine arrest $49 T lymphoma cells in the Gl phase of the cell cycle [4, 51 and deoxyadenosine might increase cyclic AMP concentrations, it is possible that cyclic AMP- and deoxyadenosine-induced cell cycle arrest are mediated by the same mechanism. To investigate this possibility, we examined the interaction of cyclic AMP and deoxyadenosine in generating cytotoxicity, the effect of deoxyadenosine on ribonucleotide reductase activity and M2 subunit messenger RNA levels, and the pattern of deoxyadenosineand cyclic AMP-induced cell cycle arrest by cytofluorimetry. We were aided in these studies when we observed that some but not all hydroxyurea-resistant S49 cell lines are resistant to both deoxyadenosine and cyclic AMP. To explore whether AdR and CAMP cell cycle arrests are generated by the same or different mechanisms we chose cell lines with normal ribonucleotide reductase activity with and without PKA activity (wild type and KIN-) and two cell lines with similar degrees of hydroxyurea resistance and, consequently, similar AdR resistance with and without PKA activity (H-500-new and H-500-1-l). In addition, we used a previously characterized AdR-resistant cell line with a mutation in the allosteric effector site for dATP on ribonucleotide reductase but which possesses normal PKA activity (dGuo-200-1). Using these cell lines we compared and contrasted cyclic AMP- and deoxyadenosine-induced cell cycle arrest. The results of these experiments suggest that deoxyadenosineand cyclic AMP-induced cell cycle arrest are probably mediated by different mechanisms. MATERIALS

AND METHODS

Materialrr. [U-“C]CDP (340 mCi/mmol) was purchased from New England Nuclear (Boston, MA). a-L-Rhamnose is a product of Eastman-Kodak (Rochester, NY). Dowex AG l-X8 and protein assay reagents were purchased from Bio-Rad (Richmond, CA). All other reagents were of the highest grade available commercially. Cell lines. All wild type and mutant S49 cells used were grown and characterized as previously described [5, 8,22, 231. The hydroxyurea cell line 500-l-l is a clone of a hydroxyurea-resistant parental line which was selected by stepwise incremental increases in hydroxyurea concentrations from 50 to 500 PM. This clone is amplified in the gene coding for the M2 subunit of ribonucleotide reductase and retains resistance to hydroxyurea after more than 2 years growth in the absence of hydroxyurea. In previous studies, we reported that all subclones (including 500-l-l) of this parental cell line are PKA deficient whereas a newer amplification (HU-500-new) has PKA activity equivalent to wild type 549 cells. The KIN- S49 cell line which was origi-

ET AL. nally selected for cyclic AMP resistance is also PKA deficient because of a mutation that results in an unstable catalytic subunit protein [24], but has wild type levels of ribonucleotide reductase activity. Cell cycle analysis. Cell suspensions were centrifuged and resuspended in a solution of cold (4°C) hypotonic staining solution (0.05 mg/ml propidium iodide in 0.1% sodium citrate and 0.1% Triton X-100) for 15 min. Cells were examined on a FACS (Becton-Dickinson, Rutherford, NJ) or an EPICS V analyzer (Coulter) equipped with a multiparameter data acquisition system (Coulter). Zsobologram analysis. Cells were cultured with varying concentrations of dibutyryl cyclic AMP and/or deoxyadenosine [in the presence of deoxycoformycin (1 g&f)-an adenosine deaminase inhibitor]. Interaction of the two agents was assessed by isobologram analysis as described by Elion, Singer, and Hitchings [25]. RNA extraction. Cells were washed in PBS and resuspended in a guanidine solution that consists of 3.5 M guanidine hydrochloride, 0.02 M potassium acetate, and 10 n&f EDTA (with a 1O:l guanidine mix to pellet volume ratio). RNA was precipitated with absolute ethanol at -20°C and then reprecipitated in guanidine and extracted with phenol:chloroform:isoamyl alcohol. All steps were carried out in solutions with diethyl pyrocarbonate (DEPC)-treated water. Dot blot. RNA was denatured in 50% deionized formamide and 6% formaldehyde at 50°C for 1 h. Samples were applied to Gene Screen Plus (New England Nuclear) nylon membranes using a dot blot (Hybri-Dot) manifold (Bethesda Research Laboratories, Gaithersburg, MD). The filter was dried and baked for 2 h at 80°C. Nick translation and probing. One to two micrograms of cDNA [26] was labeled using a nick translation reagent kit (Bethesda Research Laboratories) or an oligolabeling kit (Pharmacia, Piscataway, NJ). Hybridization was performed at 65°C overnight with salmon sperm DNA to reduce background. Dot blots were washed in 2X SSC, 1% SDS at 25°C for 30 min, and 0.1X SSC and 0.1% SDS at 65“C for 30 min and then exposed to autoradiographic film. Ribonucleotide reductase activity. Ribonucleotide reductase activity was assayed by the conversion of CDP to dCDP as previously described [ 41.

RESULTS Cyclic AMP arm! Deoxyadenosine

Are Synergistic

It is known that inhibitory agents acting at the same step in a metabolic pathway have additive effects whereas those inhibitors acting at different sites are either synergistic or antagonistic [25]. Dibutyryl cyclic AMP and deoxyadenosine in adenosine deaminase-inhibited cells are clearly synergistic (Fig. 1) in the dose ranges that span from 0 to greater than 90% cytotoxicity at 72 h. Cytotoxicity and cell cycle arrest generally parallel each other for both cyclic AMP and deoxyadenosine in wild type and mutant cell lines. For example, cyclic AMP-dependent protein kinase deficient S49 cell lines are completely resistant to both cytotoxicity and cell cycle arrest by cyclic AMP [3]. Deoxyadenosine-resistant mutant cell lines generally show more graded rather than absolute resistance but here too, the resistance to deoxyadenosine-induced cytotoxicity parallels resistance to cell cycle arrest [4].

DEOXYADENOSINE-

AND

CYCLIC

.9 8 .7

4

g

.3 .6 .5 a

CELL

CYCLE

77

ARREST

observed at low concentrations of dATP. This effect could, in turn, relate to the modest proliferative stimulus that we have observed at low concentrations of deoxyadenosine in adenosine deaminase-inhibited cells (data not shown).

1.0

o

AMP-INDUCED

The Effect of Deoxyadenosine Concentrations

::b I

.2

.3

3

.4

.6

.7

.6 .9

1.0

AdR FIG. 1. Isobologram analysis of the interaction of cyclic AMP and deoxyadenosine cytotoxicity. S49 T lymphoma cells were cultured at an initial density of 5 X 10’ cells/ml. AdR in concentrations of 1,2,5, and 10 pit4 was added in the presence of 1 PM deoxycoformytin with or without the addition of 1, 10,30,100, or 300 pM dibutyryl cyclic AMP. Cells were counted at 72 h and the results are plotted as fractional inhibitory concentrations. A straight diagonal line indicates an additive effect whereas lines above the diagonal indicate antagonism and those below the diagonal indicate synergism.

The Effect of Deoxyadenosine Reductase Activity

on Ribonucleotide

We examined the effect of deoxyadenosine toxicity on CDP reductase activity in wild type and mutant cell lines. Since ribonucleotide reductase activity is virtually absent in Gl phase cells, Gl arrest should be accompanied by a diminution in CDP reductase activity. Furthermore, if CAMP- and deoxyadenosine-induced cell cycle arrest are mediated by the same mechanism, there might be a difference in sensitivity to deoxyadenosineinduced cell cycle arrest between cells possessing normal amounts of PKA activity and those deficient in PKA. As seen in Fig. 2, KIN- cells, which are deficient in PKA activity, and wild type cells have virtually the same pattern of inhibition of CDP reductase activity by AdR. Low concentrations of AdR (10 piV) have little effect on CDP reductase activity for 6 h then a progressive decline in activity occurs to approximately onethird of control values at 24 h. Higher concentrations of deoxyadenosine result in greater inhibition of CDP reductase activity. As expected, the hydroxyurea-resistant cell lines show less sensitivity to AdR inhibition than wild type cells. However, PKA deficient hydroxyurea-resistant cell lines (HU 500 and HU-500-1-1) were no less sensitive to AdR inhibition than hydroxyurearesistant cells that possess normal PKA activity (HU500-new). The modest increase in ribonucleotide reductase activity in hydroxyurea-resistant cell lines at early time points is of unclear significance. It is possibly due to the slight increase in CDP reductase activity that can be

on M2 Messenger RNA

Recently, we reported that cyclic AMP-induced cell cycle arrest is accompanied by a decrease of messenger RNA coding for the M2 subunit of ribonucleotide reductase to undetectable levels by 6 h after cyclic AMP administration. This is coincident with the first detectable alteration in cell cycle distribution-a pronounced dip between Gl and S phase peaks observed on cytofluorographic analysis of cell cycle distribution. To determine whether the time course of the cyclic AMP effect on M2 message is similar in deoxyadenosine-treated cells we next examined the effect of deoxyadenosine toxicity on the concentration of MB-specific messenger RNA [26] using Northern dot blot analysis (Fig. 3). In wild type and KIN- cells, the MB-specific message is substantially diminished at 24 h in cells treated with 10 pM or greater concentrations of AdR. Shorter time periods of exposure to higher concentrations can also result in diminished M2 message as seen in wild type and KIN- cells treated with 100 PM AdR for 12 h. Dot blot autoradiogram intensity parallels CDP reductase activity (data not shown). Both hydroxyurea-resistant cell lines show resistance to AdR-induced diminution of MB-specific message concentrations. At 10 pM AdR for 24 h

H” 500-I-1

KIN-

8aQ 60

40

-

0 I

6

12 24

\

5 I

6

12 24

I

6

12 24

I,,, I 6

12 24

/I,, I 6

L 12 24

HOURS

FIG. 2. The effect of deoxyadenosine on ribonucleotide reductase activity. Deoxyadenosine at 10, 30, and 100 PM was added to cultures of wild type, KIN-, and three hydroxyurea-resistant cell lines. HU-500-new has normal PKA activity whereas both the parental cell line, HU-500, and a subclone, HU-500-l-1, lack detectable PKA activity. CDP reductase activity in permeabilizedcells is plotted as percentage of control after 1,6,12, and 24 h of incubation.

78 a

WT

r------AdR

h

24 h

b

(pbl) I

li

Kin-

-AdR

(PM) -

A

n

-

24 h

12 h

12 h

6h

6h

h

h

I

lh

C

lh

HU 500 NEW 1

d

AdR Q.1t.4)I 10

30

100

C

10

24 h

24 h

6h

MU 500-1-I -AdR

I,

QM) 30

A I

a

C

h

100

DEOXYADENOSINE-

AND

CYCLIC

AMP-INDUCED

substantial MB-specific mRNA is present in both hydroxyurea-resistant cell lines but not in wild type or KIN- cell lines. In summary, the M2-specific messenger RNA concentrations parallel the cell cycle distribution, with low concentrations in cells arrested in Gl by either dibutyryl cyclic AMP or deoxyadenosine and normal concentrations in cells not arrested. However, cyclic AMPinduced cell cycle arrest results in undetectable message concentration by 6 h [27] whereas the earliest reduction in M2 message by deoxyadenosine is at 12 h. Furthermore, the effect of AdR on the M2-specific message is independent of PKA activity in the four cell lines tested. Lastly, dGuo-200-1 is a cell line resistant to AdR-induced Gl arrest and cytotoxicity [4] (wild type E&,, 3.5 /.&; H-500 EC&,,,7 pM; dGUO-200-1 EC,, >20 PM). It has an altered response to dATP because of a mutation in the allosteric binding site on the Ml subunit [28-301. dGuo-200-l has normal PKA activity and response to cyclic AMP. Thus, resistance to AdR does not affect the response to cyclic AMP. Gl-arrested wild type and KIN- cells exposed to 10 pM AdR for 24 h still retain one-third of their CDP reductase activity. Retention of CDP reductase activity during AdR cell cycle arrest is even more pronounced in hydroxyurea-resistant cell lines. By contrast, cyclic AMP-arrested cells exhibit no detectable CDP reductase activity nor do they have detectable messenger RNA for M2 [27]. At least one possible explanation of the difference in CDP reductase activity and message concentration is the observation that cyclic AMP appears to arrest S49 cells in Gl at an earlier point than AdR (Fig. 4). Reversal of AdR Toxicity

by CdR

Deoxycytidine can prevent the toxicity of AdR in S49 cells, in part, by competition for phosphorylation [3133] by deoxycytidine kinase [34] (the primary enzyme responsible for pbosphorylation of deoxyadenosine). We examined this effect on cell cycle distribution and M2-specific messenger RNA concentrations in wild type cells and a hydroxyurea-resistant cell line, HU 500-l-l (Fig. 5). Although the M2-specific messenger RNA concentrations are greater in hydroxyurea-resistant cells than in wild type cells, the effect of AdR on message concentrations is the same in both cell types. Furthermore, it is clear that deoxycytidine restores exponential cell cycle distribution with consequent increase in MB-specific mRNA concentration. This pre-

CELL

CYCLE

ARREST

79

FIG. 4. Cyclic AMP and deoxyadenosine-induced Gl arrest. Wild type S49 cells were exposed to dibutyryl cyclic AMP (1 m&f) or deoxyadenosine (10 pM in the presence of the adenosine deaminase inhibitor deoxycoformycin-1 rM) for 18 h. Cytofluorographs of propidium iodide-stained cells demonstrate early Gl arrest for cyclic AMP-treated cells (left peak-dotted line) versus late Gl arrest for deoxyadenosine (right peak-solid line).

vention of arrest and toxicity by CdR is not seen in CdR-treated cyclic AMP-arrested cells. DISCUSSION

To summarize our findings, first, it is clear from the isobologram analysis that cyclic AMP and deoxyadenosine are synergistic for cytotoxicity. This implies that they operate through different mechanisms. Second, both mutant and wild type S49 cells show graded inhibition to increasing concentrations of deoxyadenosine whereas the cytotoxicity of cyclic AMP is present or absent depending on the presence of PKA activity in the cell line. Furthermore, PKA status plays no role in sensitivity to deoxyadenosine toxicity, again suggesting that they operate through different pathways. Third, while both cyclic AMP and deoxyadenosine arrest cells in Gl, cyclic AMP-induced cell cycle arrest is entirely reversible for at least 24 h whereas deoxyadenosine cell cycle arrest is irreversible and lethal. Fourth, the kinetics of inhibition of ribonucleotide reductase activity, which is present in S and G2/M phase cells, is different in deoxyadenosine- versus cyclic AMP-arrested cells. Cyclic AMP (1 mM) diminishes

FIG. 3. The effect of deoxyadenosine on Ma-specific messenger RNA concentrations. Total cellular RNA was extracted from cells cultured in the presence of AdR at varying concentrations (with 1 a deoxycoformycin) for the times indicated RNA (10 pg) was spotted on nylon filters and probed with a full length cDNA. Dot blots are shown with corresponding cytofluorographs for wild type (a), KIN- (b), HU-500-new (c), and HU-500-1-1 (d) cell lines.

80

ALBERT COlltfOl

WT

2o pM AdR

20 JIM AdR 30 ,M CdR

10 #A AdR

10 JIM AdR 30 JIM CdR

k il

FIG. 5. The effect of deoxycytidine on MZ-specific message and cell cycle distribution in deoxyadenosine-treated cells. Wild type and HU-500-1-1 were incubated for 24 h with AdR and CdR as indicated. Ma-specific messenger RNA concentrations and cytofluorographs were performed as described above.

the message for the M2 subunit by 6 h after administration. CDP reductase activity in cyclic AMP-treated cells parallels M2 message concentration. In most deoxyadenosine-treated cells there is observable M2-specific message up to 12 h after deoxyadenosine addition. Even in those cells arrested in Gl phase there is retention of up to one-third of the CDP reductase activity. These observations are consistent with the hypothesis that Gl phase cell cycle arrest induced by deoxyadenosine is later than that induced by cyclic AMP. Fifth, hydroxyurea resistance partially protects against deoxyadenosine cell cycle arrest and cytotoxicity whereas it has no direct bearing on the effect of cyclic AMP. Furthermore, there are several cell lines selected for deoxyadenosine resistance, such as those with mutated allosteric binding sites on the Ml subunit of ribonucleotide reductase, and those cell lines with diminished deoxycytidine kinase activity [32] which have the same response to cyclic AMP as wild type S49 cells. Last, deoxyadenosine-inducedcell cycle arrest and cytotoxicity can be obviated by deoxycytidine. This effect appears to be due in part to repletion of the pool of dCTP and, in part, to diminished accumulation of dATP because of competition for phosphorylation by deoxycytidine kinase and perhaps other mechanisms. However, deoxycytidine will not prevent cyclic AMPinduced cell cycle arrest or cytotoxicity. In conclusion, there appear to be separate and distinct causes of Gl cell cycle arrest induced by cyclic AMP and deoxyadenosine in S49 T lymphoma cells. We originally hypothesized that the mechanism of cell cycle arrest induced by cyclic AMP might be the

ET AL.

phosphorylation and inhibition of the M2 subunit of ribonucleotide reductase by PKA. Against this hypothesis are the observations that other inhibitors of ribonucleotide reductase act in S phase. Our more recent studies [ 351 suggest that ribonucleotide reductase inhibition by cyclic AMP can occur at concentrations of cyclic AMP insufficient to generate Gl cell cycle arrest. It is likely then that some hydroxyurea-resistant cell lines lose PKA activity to augment ribonucleotide reductase activity. In fact, we have recently observed that a PKA positive hydroxyurea-resistant cell line spontaneously lost PKA activity (while maintaining hydroxyurea resistance) 2 years after selection in hydroxyurea. This suggests that hydroxyurea treatment of S49 cells is a selective pressure, albeit indirect, against PKA activity. Thus, it appears that the failure of hydroxyurea-resistant cell lines to arrest in the Gl phase of the cell cycle when exposed to CAMP is a byproduct of the PKA negative phenotype but not the apparent reason why they acquired this phenotype. While these studies do not directly shed light on the mechanism of either deoxyadenosine- or cyclic AMPinduced cell cycle arrest and cytotoxicity they do clearly indicate that they are two separate phenomena. We gratefully acknowledge the gift of the M2 probe from Dr. Lars Thelander. Cytofluorograph analysis was performed by John Hartley (University of Chicago) and Karen Hagen (University of Illinois). Support for these investigations was provided from the National Institute ,of Health, Grant PHS ROl CA46607.

REFERENCES 1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13. 14. 15.

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AND CYCLIC AMP-INDUCED

16. Siaw, M. F. E., Mitchell, B. S., Koller, C. A., Coleman, M. S., and Hutton, J. J. (1980) Proc. Natl. Ad. Sci. USA 77,6157-6161. 17. Carson, D. A., Kaye, J., and Seegmiller, J. E. (1976) J. Clin. Invest. 67,274. 18. Carson, D. A., Kaye, J., and Seegmiller, J. E. (1976) J. Clin. Invest. 67, 274. 19. Zenser, T. V. (1975) Biochem. Biophys. Acta 404,202213. 20. Akerblom, L., Ehrenberg, A., Graslund, A., Lankinen, H., Reichard, P., and Thelander, L. (1987) Proc. N&Z. Acad. Sci. USA 78,2159-2163. 21. Eriksson, S., Graslund, A., Skog, S., Thelander, L., and Tribukait, B. (1984) J. Biol. Chem. 259, 11,695-11,700. 22. Albert, D. A., Gudas, L. J., and Nodzenski, E. (1987) J. Cell. Physiol. 130.262-269. 23. Albert, D. A., and Gudas, L. J. (1985) J. Biol. Chem. 260,679684. 24. Orellana, S. A., and McKnight, G. S. (1990) J. Biol. Chem. 265, 3048-3053. 25. Elion, G. B., Singer, S., and Hitchings, G. H. (1954) J. Bid. Chem. 208,477-488. Received June 7, 1991

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26. Thelander, L., and Berg, P. (1986) Mol. Cell. Biol. 6,3433-3442. 27. Albert, D. A., Nodzenski, E., Yim, G., and Kowalski, J. (1990) J. Cell Physiol. 143,251-256. 28. Eriksson, S., Gudas, L. J., Cliff, S. M., Caras, I., Ullman, B., and Martin, D. W., Jr. (1981) J. Bid. Chem. 256, 10,193-10,197. 29. Ullman, B., Clift, S. M., Gudas, L. J., Levinson, B. B., Wormsted, M. A., and Martin, D. W., Jr. (1980) J. Biol. Chem. 255,8303-8314. 30. Eriksson, S., Gudas, L. J., Ullman, B., Clift, S. M., and Martin, D. W., Jr. (1981) J. Biol. Chem. 256, 10,184-10,188. 31. Ullman, B., Gudas, L. J., Cohen, A., and Martin, D. W., Jr. (1978) Cell 14, 365-375. 32. Hershfield, M. S., Fetter, J. E., Small, W. C., et al. (1982) J. Biol. Chem. 257,6380-6386. 33. Mitchell, B. S., Mejias, C., Daddona, P., and Kelley, W. N. (1978) Proc. Natl. Ad. Sci. USA 75,5011-5014. 34. Ullman, B., Levinson, B. B., Hershfield, M. S., and Martin, D. W., Jr. (1981) J. Biol. Chem. 256,84&-852. 35. Albert, D. A., Kowalski, J., Nodzenski, E., Micek, M., and Wu, P. (1990) Biochem. Biophys. Res. Commun. 167, 383-390.

Deoxyadenosine- and cyclic AMP-induced cell cycle arrest and cytotoxicity.

We compared deoxyadenosine (AdR)- and cyclic AMP (cAMP)-induced cell cycle arrest and cytotoxicity in wild type and mutant S49 cells to determine whet...
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