JOURNAL OF CELLULAR PHYSIOLOGY 143251-256 (19901
Effect of Cyclic AMP on the Cell Cycle Regulation of Ribonucleotide Reductase M2 Subunit Messenger RNA Concentrations in Wild-Type and Mutant S49 T Lymphoma Cells DANIEL A. ALBERT,* EDWARDINE NODZENSKI, GLORIA YIM, AND JANICE KOWALSKI Rheumatology Section, Department of Medicine, University of Chicago, Chicago, lllinois 60637
Ribonucleotide reductase activity in S49 T lymphoma cells is cell cycle regulated by de novo protein synthesis of the M2 subunit. There is maximal enzyme activity in S and C2/M phase with low activity and low concentrations of the M2 subunit in GI phase. Pharmacologic concentrations of cyclic AMP arrest 549 cells in the G1 phase of the cell cycle. We investigated t h e effect of cyclic AMP on M2 messenger RNA concentrations using R N A from exponentially growing and elutriated, cell cycle-enriched populations. To discern whether cyclic AMP-induced G1 arrest was associated with low concentrations of M2-specific messenger RNA, we probed blots with a full-length cDNA for M2. Cell cycle variation in M2 messenger RNA concentrations was similar in wild-type, hydroxyurea-resistant cells with amplified M2 activity, and cyclic AMP-dependent protein kinase-deficient cell lines. All lines had low amounts of M2-specific mRNA in early G1, an increase at the late Gl/early S phase interface, a decrease in mid S phase, and another increase in late S phase that continued through G2/M. These concentrations did not directly correlate with enzyme activity, suggesting other regulatory effects might participate in determining ribonucleotide reductase activity. Cyclic AMP exposure appeared to induce cell cycle arrest in early G1 with low M2specific messenger R N A concentration. This eftect reversed upon washout of the cyclic AMP and was dependent on functional cyclic AMP-dependent protein kinase (PKA). These results suggest that cyclic AMP arrests S49 mouse T lymphoma cells in early G1 prior to transcriptional activation of the M2 gene. Cyclic AMP is a regulator of cell growth and proliferation (Gottesman and Fleischmann, 1986). Pharmacologic concentrations of cyclic AMP reversibly arrest S49 T lymphoma cells in the G1 phase of the cell cycle (Coffino et al., 1975a), a n effect mimicked by agents (such as theophylline and forskolin) that increase endogenous concentrations of cyclic AMP. Cyclic AMPinduced cell cycle arrest requires cyclic AMP-dependent protein kinase (protein kinase A; PKA), since mutant cells lines deficient in this enzyme resist the cell cycle arrest effect (Coffino et al., 1975b; Daniel et al., 1973; Wetters et al., 1983). Target proteins for the PKA reaction are largely unknown but include PKA itself (Russell and Steinberg, 1987), as well as others that have been identified by mobility on two-dimensional gel electrophoresis (Steinberg and Kiss, 1985) and probably the M2 subunit of ribonucleotide reductase (Albert and Nodzenski, 1989). However, neither the specific protein or proteins involved in cell cycle arrest nor the mechanism of cell cycle arrest have been identified. Ribonucleotide reductase, a n enzyme essential for DNA synthesis, catalyzes the reduction of all four ric
1990 WILEY-LISS, INC
bonucleoside diphosphates to their respective deoxyribonucleoside diphosphates, which are further phosphorylated prior to incorporation into DNA (Thelander and Reichard, 1979). The enzyme is composed of two distinct subunits: the M1 subunit is responsible for complex allosteric feedback regulation of the enzyme activity, and the M2 subunit contains tyrosyl radicals necessary for catalysis (Reichard and Ehrenberg, 1983). Hydroxyurea, a specific inhibitor of ribonucleotide reductase, binds and inactivates the tyrosyl radicals a t the catalytic site on the M2 subunit (Akerblom et al., 1981). Exposure of S49 cells to incrementally increasing concentrations of hydroxyurea results in hydroxyurea resistance, primarily as a consequence of amplification of the gene coding for the M2 subunit of ribonucleotide reductase (Choy et al., 1988). Hydroxyurea-resistant cell lines have increased ribonucleotide reductase activity, increased deoxynucleotide pools,
Received November 9, 1989; accepted January 10, 1990.
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and resistance to deoxyadenosine (Albert et al., 1987; Albert and Nodzenski, 1988). We observed that some, but not all, of our hydroxyurea-resistant cell lines were resistant to cyclic AMPinduced cell cycle arrest, and that this resistance correlates with the apparent mutational loss of PKA activity (Albert and Nodzenski, 1989).We thus hypothesized that phosphorylation of the M2 subunit by cyclic AMP-dependent protein kinase was a mechanism of inhibition of ribonucleotide reductase and that mutational loss of PKA permitted increased resistance to hydroxyurea. This hypothesis was supported by the findings that PKA-deficient, cyclic AMP-resistant cell lines not selected for hydroxyurea resistance showed threefold increased hydroxyurea resistance and that the M2, but not the M1, subunit of ribonucleotide reductase was a target for cyclic AMP-dependent protein kinase in vitro and in vivo. Under conditions of in vivo phosphorylation of M2, in fact, ribonucleotide reductase activity was diminished (Albert and Nodzenski, 1989). Several lines of evidence suggest that phosphorylation of the M2 subunit of ribonucleotide reductase by protein kinase A is unlikely to be the major mechanism of cell cycle arrest by cyclic AMP. First, ribonucleotide reductase is a n S phase-specific enzyme, and cyclic AMP arrests cells in G1. Second, protein kinase A-deficient cell lines have normal cell cycle regulation of ribonucleotide reductase activity, and therefore, enzyme activity can be regulated normally independent of phosphorylation. Third, hydroxyurea-resistant cell lines are not all cyclic AMP resistant, so t h a t simple incremental increases in M2 concentration cannot readily account for diminished cyclic AMP sensitivity. We wished, therefore, to evaluate the possibility that an effect of cyclic AMP on the transcription of ribonucleotide reductase mRNA during traverse of the cell cycle might constitute a mechanism for cyclic AMPinduced G1 phase arrest. The experiments presented here document pronounced changes in ribonucleotide reductase M2 messenger RNA concentrations during the cell cycle, and a n effect of cyclic AMP treatment on ribonucleotide reductase M2 subunit mRNA concentrations under conditions associated with G1 phase cell cycle arrest. MATERIALS AND METHODS Cell culture All wild-type and mutant S49 cells used were grown and characterized a s previously described (Albert and Gudas, 1985; Albert et al., 1987; Albert and Nodzenski, 1989). The hydroxyurea cell line 500-1-1 is a clone of a hydroxyurea-resistant parental line which was selected by stepwise incremental increases in hydroxyurea concentrations from 50 p M to 500 p M .This clone retains resistance to hydroxyurea after 2 years' growth in the absence of hydroxyurea. In previous studies (Albert and Nodzenski, 1989) we reported that all subclones (including 500-1-1) of this parental cell line are PKA deficient, whereas a newer amplification (HYU500-new) has PKA activity equivalent to wild-type S49 cells. The KIN- S49 cell line which was selected for cyclic AMP resistance is also PKA deficient but has wild-type levels of ribonucleotide reductase activity.
Centrifugal elutriation The elutriation system consists of a Beckman 52-21 Centrifuge, a JE-6 elutriation rotor with standard separation chambers, and a Masterflex pump. A sample of 4 x lo8 cells in Dulbecco's modified Eagle's media with 10% horse serum was loaded at 9.5 ml/min a t a rotor speed of 2,000 rpm. Fractions of 50-100 ml were removed a s the flow rate was increased from 11.5 mlimin to 32 mlimin (Albert et al., 1987). RNA extraction Cells were washed in PBS and resuspended in a guanidine solution t h a t consists of 3.5 M guanidine hydrochloride, 0.02 M potassium acetate, and 10 mM EDTA (with a 1 O : l guanidine mix to pellet volume ratio). RNA was precipitated with absolute ethanol a t -2O"C, and then reprecipitated in guanidine and extracted with pheno1:chloroform:isoamyalcohol(Maniatis et al., 1982). All steps were carried out in solutions with DEPC-treated water. Dot blot RNA was denatured in 50% deionized formamide and 6% formaldehyde a t 50°C for 1 hour. Samples were applied to Gene Screen Plus (New England Nuclear, Boston, MA) nylon membranes using a dot blot (HybriDot) manifold (Bethesda Research Laboratories, Gaithersburg, MD) (Kafatos et al., 1979). The filter was dried and baked for 2 hours at 80°C. Nick translation and probing One to 2 pg of cDNA (Thelander and Berg, 1986) was nick translated using a Nick Translation Reagent Kit (Bethesda Research Laboratories, Gaithersburg, MD) or a n oligolabeling kit (Pharmacia, Piscataway, NJ). Hybridization was performed at 65°C overnight with salmon sperm DNA to reduce background. Dot blots were washed in 2 x SSC with 1% SDS a t 65°C for 30 minutes and in 0.1 x SSC a t 25°C for 30 minutes and then exposed to autoradiographic film. N o r t h e r n blot RNA (10 pg) was denatured as described above and electrophoresed through a 1% formaldehyde-formamide agarose gel transferred to nylon filter paper (Gene Screen Plus) by capillary action and probed as described above, except that blots were washed in 2 x SSPE .1%SDS a t 25°C for 30 minutes then for 60 minutes a t 50°C. Ribonucleotide reductase Ribonucleotide reductase activity was assayed by the conversion of CDP to dCDP as previously described (Albert et al., 1987). RESULTS Cell cycle variation in M2 messenger RNA concentration and ribonucleotide reductase enzyme activity Our previous investigations have documented cell cycle variation in ribonucleotide reductase activity (Albert and Gudas, 1985). In wild-type S49 cells, enzyme activity is low in G1, higher in S phase and, in G2iM phase, is either as high or higher than in S phase. Low
EFFECT OF CYCLIC AMP
50 1
400
Wild Type Early G1
Early
S
S Phase
G21M
HYU-R Early
G1
Early S
S
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Fig. 1. M2-specific messenger RNA concentrations and CDP reductase activity during the cell cycle of S49 cells. Exponentially growing and cell cycle-specific populations were obtained by centrifugal elutriation of wild-type and hydroxyurea-resistant (500-1-1) cell types. The cell cycle distribution is shown in cytofluorographs. The u p p e r g r a p h displays the CDP reductase activity in elutriated fractions of wild-type cells that represents sequential stages during the cell cycle with exponentially growing cells shown as a single point on the left. M2-specific messenger RNA concentrations are shown as dot blots of 10 )*g total cellular RNA from the fraction shown above each dot probed with a full-length mouse M2 cDNA. Dot intensity is demonstrated by autoradiography and is proportional to amount of Ma-specific message. Wild-type and hydroxyurea-resistant cells have the same cell cycle pattern, although in cell cycle-specific fractions greater intensity of the hydroxyurea resistant dots is apparent. Furthermore, wild-type cells do not demonstrate correlation of M2 message concentration with enzyme activity.
levels of enzyme activity occur in G1 whether the cells are selected by centrifugal elutriation or by cyclic AMP-induced cell cycle arrest. A similar pattern of cell cycle variation was observed in hydroxyurea-resistant mutants with increased enzyme activity which appeared to be predominantly due to increased M2 activity (Albert et al., 1987). Other investigations (Eriksson et al., 1984; Engstrom et al., 1985) have demonstrated that the major source of cell cycle variation is increased M2 activity in S and GBIM phase. Thus, we explored M2 messenger RNA concentrations in cell cycle-specific populations of wild-type and mutant cell lines. Figure 1 shows dot blot quantitation and cytofluorometric patterns from wild-type and from one hydroxyurea-resistant clonal cell line (500-1-1) when cell cycle-specific populations were obtained by centrifugal
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elutriation. Ten microgram samples of total cellular RNA per dot were probed with a full-length cDNA for mouse M2 (courtesy of Dr. L. Thelander). It is clear that cells approximately tenfold resistant to hydroxyurea with a fourfold increase in enzyme activity (Albert et al., 1987) have a substantial increase in the messenger RNA concentration. However, the pattern of messenger RNA concentrations specific for M2 appears to be similar between wild-type and hydroxyurearesistant (HYU-R) mutants and consists of a low amount in early G1, a rise in the interface between late G1 and early S phase, a diminution in mid S phase and subsequent rise in later S phase and G2IM. Exponential cell fractions (not shown) appear to be an average of the cell cycle-specific concentrations. Furthermore, enzyme activity (Fig. 1) does not correlate with the messenger RNA concentration; thus, there appear to be other determinants of enzyme activity besides M2 message concentration. Wild-type and PKA-deficient cell lines h a v e similar cell cycle variation in Ma-specific message concentration To explore the effect of cyclic AMP on messenger RNA concentrations we first needed to ascertain the pattern of specific message for M2 in cyclic AMP-dependent protein kinase-deficient cell lines. We observed that wild-type cells and the KIN- cell line, which has deficient cyclic AMP-dependent protein kinase (PKA) activity, have largely the same cell cycle variation in messenger RNA concentration for M2. We obtained more fractions from these elutriations (not shown), and this more detailed study demonstrates a narrow zone of increased concentration a t the GUS phase interface in both cell lines. Cyclic A M P arrests wild-type but not PKA-deficient cell lines The similarity of KIN- cells to wild-type allowed us to compare the effect of cyclic AMP on messenger RNA concentration in wild-type and KIN- cell lines. As shown in Figure 2, wild-type exponentially growing cells exposed to cyclic AMP for 24 hours arrest in G1 phase with a low level of messenger RNA specific for M2. This suggests that the cells are arrested in early G1 phase rather than late G1. As shown in the bottom of that figure, cyclic AMP-treated KIN- cells have the same cell cycle distribution as exponentially growing KIN- cells and approximately the same messenger RNA concentration. These cyclic AMP-treated KIN cells also show the previously described pattern of variation of M2 messenger RNA concentrations when they are elutriated a s demonstrated by the low M2 specific messenger RNA concentration in G1 elutriated cells. ~
Cyclic A M P reversibly arrests 549 cells in e a r l y G1 with low Ma-specific messenger RNA concentrations We wished to correlate M2-specific messenger RNA concentration with cell cycle distribution in cyclic AMP-treated cells as they are arrested in G1, and subsequently released. Exponentially growing wild-type cells were exposed to 1 mM dibutyryl cyclic AMP and aliquots were taken out a t 1, 2, 4, 6 , 12, 18, and 24 hours. The cytofluorographs and messenger RNA con-
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the dot blot findings and determine which of the two RNA species for M2 (2.1 kb and 1.6 kb; as previously described by Thelander and Berg, 1986) correlate with dot blot analysis. As shown (Fig. 4) the 2.1 kb transcript from wild-type and KIN HYU-resistant cell lines diminishes with cyclic AMP treatment, but the 2.1 kb band in KIN cell lines whose cell cycle distribution is unaffected by cyclic AMP is unchanged. The 1.6 kb transcript, best seen in the HYU-resistant KIN+ cell line below the 2.1 kb band, also may diminish with cyclic AMP treatment. The band at the top of the autoradiogram which is the same intensity in all the lanes, migrates at 28s. The moderate intensity band just below the 2 8 s band in the HYU-resistant KIN ' lane is of unknown significance.
Wild Type EXP
cAMP
+
KINEarly EXP
cAMP
GI
Fig. 2. Cyclic AMP arrests wild-type S49 Cells in G1 with low concentrations of M2-specific message but has no effect on KIN cells. Wild-type S49 cells were exposed to 1 mM dibutyryl cyclic AMP for 24 hours, and M2-specific message and cytofluorographs are shown. KIN cells were also exposed to 1 mM dibutyryl cyclic AMP (CAMP) for 24 hours and a r e shown next to exponentially growing KIN cells. Cell cycle-specific populations of KIN- cells shown were exposed to 1 mM dibutyryl cyclic AMP and then elutriated, and the G1-enriched fraction is shown.
centrations specific for M2 are shown Figure 3. They demonstrate that by 6 hours there is undetectable message in cyclic AMP-treated cells as the cell cycle distribution is being arrested in G1. At this point, there is a steep depression in the cleft between G1 and S phase in the cytofluorograph and most of the cells are either in early G1 or mid S phase, both of which have relatively low levels of messenger RNA for M2. The same cells, when washed out of cyclic AMP and observed a t 0 , 1 , 2 , 4 , 6 , 1 2 , 1 8 ,and 24 hours, show a delayed release from G1 arrest which begins discernably at 12 hours after washout of cyclic AMP, and is close to exponential by 24 hours. Messenger RNA concentrations for M2 are not detectable until somewhere between 12 and 18 hours after washout, suggesting that messenger RNA concentration for M2 lags somewhat behind the release of G1 arrest. N o r t h e r n blot analysis of cyclic AMP-treated cell lines We examined exponentially growing and cyclic AMP-treated cells by Northern blot analysis to confirm
DISCUSSION These experiments show a strikingly consistent pattern of cell cycle variation in messenger RNA concentration for the M2 subunit of ribonucleotide reductase. This cell cycle variation includes low levels in early G1 phase, and a very brief increase in M2 messenger RNA concentration at the GUS interphase, low levels in early to mid S phase, and a rise in later S phase and G2/M. The same basic cell cycle variation is present in hydroxyurea-resistant cells that are amplified in M2 activity, at least in part due to gene amplification (data not shown). The wild-type pattern of cell cycle variation in M2-specific messenger RNA concentration is also exhibited by cyclic AMP-dependent protein kinase-deficient cells, and by hydroxyurea-resistant cell lines both with and without cyclic AMP-dependent protein kinase activity. Thus, under normal circumstances neither PKA activity nor M2 gene amplification alters the pattern of cell cycle regulation of M2-specific message. Furthermore, M2 messenger RNA concentrations do not correlate with enzyme activity. Enzyme activity does correlate, however, with M2 protein concentration (Engstrom et al., 1985; Eriksson et al., 1984). Thus, in addition to transcriptional and post-translational regulation, it is possible that post-transcriptional regulation of ribonucleotide reductase plays a role in gene expression. This has been suggested for another DNA synthetic enzyme, thymidine kinase (Sherley and Kelly, 19881, where efficiency of translation is thought to affect gene expression. The effect of cyclic AMP on M2-specific messenger RNA concentration is dramatically different in those cells possessing cyclic AMP-dependent protein kinase activity than those that are deficient. Wild-type cells are arrested in G1 phase by cyclic AMP and cyclic AMP analogs, a s shown in Figures 2 and 3. This arrest is accompanied by a diminution in messenger RNA concentration. In cyclic AMP-dependent protein kinasedeficient cells, however, there is no effect of cyclic AMP on messenger RNA concentrations, either in exponentially growing cells that remain exponentially growing after cyclic AMP treatment, or in those cells exposed to cyclic AMP and then elutriated into cell cycle-specific fractions. The progressive decline in M2 messenger RNA concentrations in wild-type cells exposed to cyclic AMP parallels their arrest in G1 and subsequent release from G1 arrest when cyclic AMP is washed out of the cells. However, i t appears that the messenger RNA concentration lags slightly behind the initial release
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Fig. 3. Wild type S49 cells exposed to cyclic AMP reversibly arrest in GI with low M2-specific messenger RNA concentrations. Exponentially growing wild-type cells (exp) were exposed for 1, 2.4, 6, 12, 18, and 24 hours to 1 mM dibutyryl cyclic AMP. The ribonucleotide reductase activity (CDP reductase activity in percent of exponential cell
--Wild Type
Exp
CAMP
HYU Resistant HYU Resistant KIN* KINE X ~ CAMP
E X ~ CAMP
2.1 kb
Fig. 4. Northern blot analysis-10 kg total cellular RNA was electrophoresed through a formaldehyde agarose gel transferred t~ nylon filter paper then probed with the same cDNA that was used in previous experiments. The 2.1 kb RNA species was identified in wildtype, 500-new (HYU-resistant KIN') and 500-1-1 (HYU-resistant KIN-) cell lines and was increased in hydroxyurea-resistant cells that were amplified in CDP reductase activity. Only PKA-positive cells show diminution of this transcript when cells were exposed to cyclic AMP.
from G1 arrest and at least does not precede the release from G1 arrest. This suggests that there may not be a direct cause and effect relationship between the diminution in M2-specific messenger RNA concentrations and cell cycle arrest. Indeed, deoxyadenosine also induces cell cycle arrest in early G1 with low M2 specific mRNA concentrations (Albert et al., unpublished observations) probably through a different mechanism than cyclic AMP. The variation of messenger RNA concentration could come a s a result of either transcriptional regulation (Roesler et al., 1988) or changes in message stability
culture control), cell cycle distribution, and M2 message concentrations are shown in the left panel. The right panel shows the same data for cells exposed to cyclic AMP for 24 hours then washed out and assayed at 0, 1, 2, 4, 6, 12, 18, and 24 hours after washout. Each dot is shown in duplicate to demonstrate reproducibility.
(i.e., message half-life), which has been thought to decline after translation (Rosenthal et al., 1980; Brock and Shapiro, 1983). It is possible that either of these processes could result in variation of message concentration. Cyclic AMP treatment results in an accumulation of cells in G1 phase with a decline in M2-specific messenger RNA concentration and a progressive decline in ribonucleotide reductase enzyme activity, whereas cells beyond the GUS interphase proceed through the cell cycle. Thus, these data suggest that a protein phosphorylated by cyclic AMP-dependent protein kinase could act as a transcriptional regulator in S49 cells and could participate in the cell cycle arrest. This effect may be mediated by a cyclic AMP-responsive element as has been shown for cyclic AMP-induced activation of gene transcription (Montminy and Bilezikjian, 1987) but to date it has only been described as a positive effector element. The alternative hypothesis that cyclic AMP affects message stability is partially supported by the presence of a n AU-rich sequence in the 3' flanking region of messenger RNA that is thought to confer instability (Shaw and Kamen, 1986).Further experiments will be necessary to determine which of these possibilities is correct.
ACKNOWLEDGMENTS We thank L. Thelander for the gift of the plasmid containing the M2 cDNA and Michael Becker for assistance in performing the Northern blot analysis and critical review of this manuscript. Technical support was provided by J. Kuchibhotla. These studies were supported by a n Arthritis Investigator Award from the Arthritis Foundation and Public Health Service grant CA46607 from the National Cancer Institute. LITERATURE CITED Akerblom, L., Ehrenberg, A,, Graslund, H., Lankinen, H., Reichard, P., and Thelander, L. (1981) Overproduction of the free radical of ribonucleotide reductase in hydroxyurea-resistant mouse fibroblast 3T6 cells. Roc. Natl. Acad. Sci. U S A . , 78t2159-2163. Albert, D.A., and Gudas, L.J. (1985) Deoxyribonucleoside triphosphate metabolism during the cell cycle. J. Biol. Chem., 260:679684. Albert. D.A.. Gudas. L.J.. and Nodzenski. E. (1987)Deoxvribonucle-
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otide metabolism and cyclic AMP resistance in Hydroxyurea-resistant S49 T lymphoma cells. J. Cell. Physiol., 130:262-269. Albert, D.A., and Nodzenski, E. (1988) Deoxyadenosine toxicity in Hydroxyurea resistant 549 T-lymphoma cells. Exp. Cell Res., 179: 417-428. Albert, D.A., and Nodzenski, E. (1989)The M2 subunit of ribonucleotide reductase is a target of cyclic AMP dependent protein kinase. J. Cell. Physiol., 138t129-136. Brock, M.L., and Shapiro, D.J. (19831Estrogen stabilizes vitellogenen mRNA against cytoplasmic degradation. Cell, 34.207-214. Choy, B.K., McClarty, G.A., Chan, A.K., Thelander, L., and Wright, J.A. (1988) Molecular mechanisms of drug resistance involving ribonucleotide reductase: Hydroxyurea resistance in a series of clonally related cell lines selected in the presence of increasing drug concentrations. Cancer Res., 48r2029-2035. Coffino, P., Bourne, H.R., and Tomkins, G.M. (1975a) Cyclic AMP, and nonessential regulator of the cell cycle. Proc. Natl. Acad. Sci. USA, 72:878-882. Coffino, P., Bourne, H.R., and Tomkins, G.M. (1975b)Somatic genetic analysis of cyclic AMP action: Selection of unresponsive mutants. J . Cell. Physiol., 85:603-610. Daniel, V., Litwack, G., and Tomkins, G.M. (1973) Induction of cytolysis of cultured lymphoma cells by adenosine 3’5’-cyclic adenosine monophosphate and isolation of resistant variants. Proc. Natl. Acad. Sci. USA, 70:76-79. Engstrom, Y., Eriksson, S., Jildavik, I., Skog, S., Thelander, L., and Tribukait, B. (1985)Cell cycle-dependent expression of mammalian ribonucleotide reductase: Differential regulation of the two subunits. J . Biol. Chem., 260r9114-9116. Eriksson, S., Graslund, A,, Skog, S., Thelander, L., and Tribukait, B. (1984) Cell cycle-dependent regulation of mammalian ribonucleotide reductase: The S phase correlated increase in subunit M2 is regulated by de novo protein synthesis. J. Biol. Chem., 259:1169511700. Gottesman, M., and Fleischmann, R.D. (1986) The role of CAMPin regulating tumor cell growth. Cancer Surveys, 5.991-308. Kafatos, F.C., Jones, C.W., and Efstratiadis, A. (1979)A determina-
tion of nucleic acid sequence homologies and relative concentrations by a dot blot hybridization procedure. Nucleic Acids Res., 7: 1541-1552. Maniatis, T., Fritsch, E.F., and Sambrook, J. (1982) Molecular Cloning: A Laboratory Manual. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY. Montminy, M.R., and Bilezikjian, L.M. (1987) Binding of a nuclear protein to the cyclic-AMP response element of the somatostatin gene. Nature, 328:174-178. Reichard, P., and Ehrenberg, A. (1983) Ribonucleotide reductaseRadical enzyme. Science, 221.514-519. Roesler, W.J., Vandenbark, G.R., and Hanson, R.W. (1988) Cyclic AMP and the induction of eukaryotic gene transcription. J. Bid. Chem., 263:9063-9066. Rosenthal, E.T., Hunt, T., and Ruderman, J.V. (1980)Selective translation of mRNA controls the pattern of protein synthesis during development of the surf clam spicula solidissima. Cell, 2Or487-494. Russell, J.L., and Steinberg, R.A. (1987) Phosphorylation of regulatory subunit of type 1 cyclic AMP-dependent protein kinasebiphasic effects of cyclic AMP in intact 549 mouse lymphoma cells. J . Cell. Physiol., 130:203-213. Shaw, G. and Kamen, R. (1986)A conserved AU sequence from the 3’ untranslated region of GM-CSF mRNA mediates selective mRNA degradation. Cell, 46r659-667. Sherley, J.L., and Kelly, T.J. (1988) Regulation of human thymidine kinase during the cell cycle. J. Biol. Chem., 2633350-8358. Steinberg, R.A., and Kiss, Z. (1985) Basal phosphorylation of cyclic AMP-regulated phosphoproteins in intact S49 mouse lymphoma cells. Biochem. J., 227t981-994. Thelander, L., and Berg, P. (1986) Isolation and characterization of expressible cDNA clones encoding the M1 and M2 subunits of mouse ribonucleotide reductase. Mol. Cell. Biol., 6:3433-3442. Thelander, L., and Reichard, P. (1979) Reduction of ribonucleotides. Annu. Rev. Biochem., 48:133-158. Wetters, T.V.D.,Murtaugh, M.P., and Coffino, P. (19831Revertants of a transdominant S49 mouse lymphoma mutant that affects expression of a CAMP-dependent protein kinase. Cell, 35r311-320.
Effect of Cyclic AMP on the Cell Cycle Regulation of Ribonucleotide Reductase M2 Subunit Messenger RNA Concentrations in Wild-Type and Mutant S49 T Lymphoma Cells DANIEL A. ALBERT,* EDWARDINE NODZENSKI, GLORIA YIM, AND JANICE KOWALSKI Rheumatology Section, Department of Medicine, University of Chicago, Chicago, lllinois 60637
Ribonucleotide reductase activity in S49 T lymphoma cells is cell cycle regulated by de novo protein synthesis of the M2 subunit. There is maximal enzyme activity in S and C2/M phase with low activity and low concentrations of the M2 subunit in GI phase. Pharmacologic concentrations of cyclic AMP arrest 549 cells in the G1 phase of the cell cycle. We investigated t h e effect of cyclic AMP on M2 messenger RNA concentrations using R N A from exponentially growing and elutriated, cell cycle-enriched populations. To discern whether cyclic AMP-induced G1 arrest was associated with low concentrations of M2-specific messenger RNA, we probed blots with a full-length cDNA for M2. Cell cycle variation in M2 messenger RNA concentrations was similar in wild-type, hydroxyurea-resistant cells with amplified M2 activity, and cyclic AMP-dependent protein kinase-deficient cell lines. All lines had low amounts of M2-specific mRNA in early G1, an increase at the late Gl/early S phase interface, a decrease in mid S phase, and another increase in late S phase that continued through G2/M. These concentrations did not directly correlate with enzyme activity, suggesting other regulatory effects might participate in determining ribonucleotide reductase activity. Cyclic AMP exposure appeared to induce cell cycle arrest in early G1 with low M2specific messenger R N A concentration. This eftect reversed upon washout of the cyclic AMP and was dependent on functional cyclic AMP-dependent protein kinase (PKA). These results suggest that cyclic AMP arrests S49 mouse T lymphoma cells in early G1 prior to transcriptional activation of the M2 gene. Cyclic AMP is a regulator of cell growth and proliferation (Gottesman and Fleischmann, 1986). Pharmacologic concentrations of cyclic AMP reversibly arrest S49 T lymphoma cells in the G1 phase of the cell cycle (Coffino et al., 1975a), a n effect mimicked by agents (such as theophylline and forskolin) that increase endogenous concentrations of cyclic AMP. Cyclic AMPinduced cell cycle arrest requires cyclic AMP-dependent protein kinase (protein kinase A; PKA), since mutant cells lines deficient in this enzyme resist the cell cycle arrest effect (Coffino et al., 1975b; Daniel et al., 1973; Wetters et al., 1983). Target proteins for the PKA reaction are largely unknown but include PKA itself (Russell and Steinberg, 1987), as well as others that have been identified by mobility on two-dimensional gel electrophoresis (Steinberg and Kiss, 1985) and probably the M2 subunit of ribonucleotide reductase (Albert and Nodzenski, 1989). However, neither the specific protein or proteins involved in cell cycle arrest nor the mechanism of cell cycle arrest have been identified. Ribonucleotide reductase, a n enzyme essential for DNA synthesis, catalyzes the reduction of all four ric
1990 WILEY-LISS, INC
bonucleoside diphosphates to their respective deoxyribonucleoside diphosphates, which are further phosphorylated prior to incorporation into DNA (Thelander and Reichard, 1979). The enzyme is composed of two distinct subunits: the M1 subunit is responsible for complex allosteric feedback regulation of the enzyme activity, and the M2 subunit contains tyrosyl radicals necessary for catalysis (Reichard and Ehrenberg, 1983). Hydroxyurea, a specific inhibitor of ribonucleotide reductase, binds and inactivates the tyrosyl radicals a t the catalytic site on the M2 subunit (Akerblom et al., 1981). Exposure of S49 cells to incrementally increasing concentrations of hydroxyurea results in hydroxyurea resistance, primarily as a consequence of amplification of the gene coding for the M2 subunit of ribonucleotide reductase (Choy et al., 1988). Hydroxyurea-resistant cell lines have increased ribonucleotide reductase activity, increased deoxynucleotide pools,
Received November 9, 1989; accepted January 10, 1990.
‘$Towhom reprint requestsicorrespondence should be addressed.
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and resistance to deoxyadenosine (Albert et al., 1987; Albert and Nodzenski, 1988). We observed that some, but not all, of our hydroxyurea-resistant cell lines were resistant to cyclic AMPinduced cell cycle arrest, and that this resistance correlates with the apparent mutational loss of PKA activity (Albert and Nodzenski, 1989).We thus hypothesized that phosphorylation of the M2 subunit by cyclic AMP-dependent protein kinase was a mechanism of inhibition of ribonucleotide reductase and that mutational loss of PKA permitted increased resistance to hydroxyurea. This hypothesis was supported by the findings that PKA-deficient, cyclic AMP-resistant cell lines not selected for hydroxyurea resistance showed threefold increased hydroxyurea resistance and that the M2, but not the M1, subunit of ribonucleotide reductase was a target for cyclic AMP-dependent protein kinase in vitro and in vivo. Under conditions of in vivo phosphorylation of M2, in fact, ribonucleotide reductase activity was diminished (Albert and Nodzenski, 1989). Several lines of evidence suggest that phosphorylation of the M2 subunit of ribonucleotide reductase by protein kinase A is unlikely to be the major mechanism of cell cycle arrest by cyclic AMP. First, ribonucleotide reductase is a n S phase-specific enzyme, and cyclic AMP arrests cells in G1. Second, protein kinase A-deficient cell lines have normal cell cycle regulation of ribonucleotide reductase activity, and therefore, enzyme activity can be regulated normally independent of phosphorylation. Third, hydroxyurea-resistant cell lines are not all cyclic AMP resistant, so t h a t simple incremental increases in M2 concentration cannot readily account for diminished cyclic AMP sensitivity. We wished, therefore, to evaluate the possibility that an effect of cyclic AMP on the transcription of ribonucleotide reductase mRNA during traverse of the cell cycle might constitute a mechanism for cyclic AMPinduced G1 phase arrest. The experiments presented here document pronounced changes in ribonucleotide reductase M2 messenger RNA concentrations during the cell cycle, and a n effect of cyclic AMP treatment on ribonucleotide reductase M2 subunit mRNA concentrations under conditions associated with G1 phase cell cycle arrest. MATERIALS AND METHODS Cell culture All wild-type and mutant S49 cells used were grown and characterized a s previously described (Albert and Gudas, 1985; Albert et al., 1987; Albert and Nodzenski, 1989). The hydroxyurea cell line 500-1-1 is a clone of a hydroxyurea-resistant parental line which was selected by stepwise incremental increases in hydroxyurea concentrations from 50 p M to 500 p M .This clone retains resistance to hydroxyurea after 2 years' growth in the absence of hydroxyurea. In previous studies (Albert and Nodzenski, 1989) we reported that all subclones (including 500-1-1) of this parental cell line are PKA deficient, whereas a newer amplification (HYU500-new) has PKA activity equivalent to wild-type S49 cells. The KIN- S49 cell line which was selected for cyclic AMP resistance is also PKA deficient but has wild-type levels of ribonucleotide reductase activity.
Centrifugal elutriation The elutriation system consists of a Beckman 52-21 Centrifuge, a JE-6 elutriation rotor with standard separation chambers, and a Masterflex pump. A sample of 4 x lo8 cells in Dulbecco's modified Eagle's media with 10% horse serum was loaded at 9.5 ml/min a t a rotor speed of 2,000 rpm. Fractions of 50-100 ml were removed a s the flow rate was increased from 11.5 mlimin to 32 mlimin (Albert et al., 1987). RNA extraction Cells were washed in PBS and resuspended in a guanidine solution t h a t consists of 3.5 M guanidine hydrochloride, 0.02 M potassium acetate, and 10 mM EDTA (with a 1 O : l guanidine mix to pellet volume ratio). RNA was precipitated with absolute ethanol a t -2O"C, and then reprecipitated in guanidine and extracted with pheno1:chloroform:isoamyalcohol(Maniatis et al., 1982). All steps were carried out in solutions with DEPC-treated water. Dot blot RNA was denatured in 50% deionized formamide and 6% formaldehyde a t 50°C for 1 hour. Samples were applied to Gene Screen Plus (New England Nuclear, Boston, MA) nylon membranes using a dot blot (HybriDot) manifold (Bethesda Research Laboratories, Gaithersburg, MD) (Kafatos et al., 1979). The filter was dried and baked for 2 hours at 80°C. Nick translation and probing One to 2 pg of cDNA (Thelander and Berg, 1986) was nick translated using a Nick Translation Reagent Kit (Bethesda Research Laboratories, Gaithersburg, MD) or a n oligolabeling kit (Pharmacia, Piscataway, NJ). Hybridization was performed at 65°C overnight with salmon sperm DNA to reduce background. Dot blots were washed in 2 x SSC with 1% SDS a t 65°C for 30 minutes and in 0.1 x SSC a t 25°C for 30 minutes and then exposed to autoradiographic film. N o r t h e r n blot RNA (10 pg) was denatured as described above and electrophoresed through a 1% formaldehyde-formamide agarose gel transferred to nylon filter paper (Gene Screen Plus) by capillary action and probed as described above, except that blots were washed in 2 x SSPE .1%SDS a t 25°C for 30 minutes then for 60 minutes a t 50°C. Ribonucleotide reductase Ribonucleotide reductase activity was assayed by the conversion of CDP to dCDP as previously described (Albert et al., 1987). RESULTS Cell cycle variation in M2 messenger RNA concentration and ribonucleotide reductase enzyme activity Our previous investigations have documented cell cycle variation in ribonucleotide reductase activity (Albert and Gudas, 1985). In wild-type S49 cells, enzyme activity is low in G1, higher in S phase and, in G2iM phase, is either as high or higher than in S phase. Low
EFFECT OF CYCLIC AMP
50 1
400
Wild Type Early G1
Early
S
S Phase
G21M
HYU-R Early
G1
Early S
S
Phase
G21M
Fig. 1. M2-specific messenger RNA concentrations and CDP reductase activity during the cell cycle of S49 cells. Exponentially growing and cell cycle-specific populations were obtained by centrifugal elutriation of wild-type and hydroxyurea-resistant (500-1-1) cell types. The cell cycle distribution is shown in cytofluorographs. The u p p e r g r a p h displays the CDP reductase activity in elutriated fractions of wild-type cells that represents sequential stages during the cell cycle with exponentially growing cells shown as a single point on the left. M2-specific messenger RNA concentrations are shown as dot blots of 10 )*g total cellular RNA from the fraction shown above each dot probed with a full-length mouse M2 cDNA. Dot intensity is demonstrated by autoradiography and is proportional to amount of Ma-specific message. Wild-type and hydroxyurea-resistant cells have the same cell cycle pattern, although in cell cycle-specific fractions greater intensity of the hydroxyurea resistant dots is apparent. Furthermore, wild-type cells do not demonstrate correlation of M2 message concentration with enzyme activity.
levels of enzyme activity occur in G1 whether the cells are selected by centrifugal elutriation or by cyclic AMP-induced cell cycle arrest. A similar pattern of cell cycle variation was observed in hydroxyurea-resistant mutants with increased enzyme activity which appeared to be predominantly due to increased M2 activity (Albert et al., 1987). Other investigations (Eriksson et al., 1984; Engstrom et al., 1985) have demonstrated that the major source of cell cycle variation is increased M2 activity in S and GBIM phase. Thus, we explored M2 messenger RNA concentrations in cell cycle-specific populations of wild-type and mutant cell lines. Figure 1 shows dot blot quantitation and cytofluorometric patterns from wild-type and from one hydroxyurea-resistant clonal cell line (500-1-1) when cell cycle-specific populations were obtained by centrifugal
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elutriation. Ten microgram samples of total cellular RNA per dot were probed with a full-length cDNA for mouse M2 (courtesy of Dr. L. Thelander). It is clear that cells approximately tenfold resistant to hydroxyurea with a fourfold increase in enzyme activity (Albert et al., 1987) have a substantial increase in the messenger RNA concentration. However, the pattern of messenger RNA concentrations specific for M2 appears to be similar between wild-type and hydroxyurearesistant (HYU-R) mutants and consists of a low amount in early G1, a rise in the interface between late G1 and early S phase, a diminution in mid S phase and subsequent rise in later S phase and G2IM. Exponential cell fractions (not shown) appear to be an average of the cell cycle-specific concentrations. Furthermore, enzyme activity (Fig. 1) does not correlate with the messenger RNA concentration; thus, there appear to be other determinants of enzyme activity besides M2 message concentration. Wild-type and PKA-deficient cell lines h a v e similar cell cycle variation in Ma-specific message concentration To explore the effect of cyclic AMP on messenger RNA concentrations we first needed to ascertain the pattern of specific message for M2 in cyclic AMP-dependent protein kinase-deficient cell lines. We observed that wild-type cells and the KIN- cell line, which has deficient cyclic AMP-dependent protein kinase (PKA) activity, have largely the same cell cycle variation in messenger RNA concentration for M2. We obtained more fractions from these elutriations (not shown), and this more detailed study demonstrates a narrow zone of increased concentration a t the GUS phase interface in both cell lines. Cyclic A M P arrests wild-type but not PKA-deficient cell lines The similarity of KIN- cells to wild-type allowed us to compare the effect of cyclic AMP on messenger RNA concentration in wild-type and KIN- cell lines. As shown in Figure 2, wild-type exponentially growing cells exposed to cyclic AMP for 24 hours arrest in G1 phase with a low level of messenger RNA specific for M2. This suggests that the cells are arrested in early G1 phase rather than late G1. As shown in the bottom of that figure, cyclic AMP-treated KIN- cells have the same cell cycle distribution as exponentially growing KIN- cells and approximately the same messenger RNA concentration. These cyclic AMP-treated KIN cells also show the previously described pattern of variation of M2 messenger RNA concentrations when they are elutriated a s demonstrated by the low M2 specific messenger RNA concentration in G1 elutriated cells. ~
Cyclic A M P reversibly arrests 549 cells in e a r l y G1 with low Ma-specific messenger RNA concentrations We wished to correlate M2-specific messenger RNA concentration with cell cycle distribution in cyclic AMP-treated cells as they are arrested in G1, and subsequently released. Exponentially growing wild-type cells were exposed to 1 mM dibutyryl cyclic AMP and aliquots were taken out a t 1, 2, 4, 6 , 12, 18, and 24 hours. The cytofluorographs and messenger RNA con-
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the dot blot findings and determine which of the two RNA species for M2 (2.1 kb and 1.6 kb; as previously described by Thelander and Berg, 1986) correlate with dot blot analysis. As shown (Fig. 4) the 2.1 kb transcript from wild-type and KIN HYU-resistant cell lines diminishes with cyclic AMP treatment, but the 2.1 kb band in KIN cell lines whose cell cycle distribution is unaffected by cyclic AMP is unchanged. The 1.6 kb transcript, best seen in the HYU-resistant KIN+ cell line below the 2.1 kb band, also may diminish with cyclic AMP treatment. The band at the top of the autoradiogram which is the same intensity in all the lanes, migrates at 28s. The moderate intensity band just below the 2 8 s band in the HYU-resistant KIN ' lane is of unknown significance.
Wild Type EXP
cAMP
+
KINEarly EXP
cAMP
GI
Fig. 2. Cyclic AMP arrests wild-type S49 Cells in G1 with low concentrations of M2-specific message but has no effect on KIN cells. Wild-type S49 cells were exposed to 1 mM dibutyryl cyclic AMP for 24 hours, and M2-specific message and cytofluorographs are shown. KIN cells were also exposed to 1 mM dibutyryl cyclic AMP (CAMP) for 24 hours and a r e shown next to exponentially growing KIN cells. Cell cycle-specific populations of KIN- cells shown were exposed to 1 mM dibutyryl cyclic AMP and then elutriated, and the G1-enriched fraction is shown.
centrations specific for M2 are shown Figure 3. They demonstrate that by 6 hours there is undetectable message in cyclic AMP-treated cells as the cell cycle distribution is being arrested in G1. At this point, there is a steep depression in the cleft between G1 and S phase in the cytofluorograph and most of the cells are either in early G1 or mid S phase, both of which have relatively low levels of messenger RNA for M2. The same cells, when washed out of cyclic AMP and observed a t 0 , 1 , 2 , 4 , 6 , 1 2 , 1 8 ,and 24 hours, show a delayed release from G1 arrest which begins discernably at 12 hours after washout of cyclic AMP, and is close to exponential by 24 hours. Messenger RNA concentrations for M2 are not detectable until somewhere between 12 and 18 hours after washout, suggesting that messenger RNA concentration for M2 lags somewhat behind the release of G1 arrest. N o r t h e r n blot analysis of cyclic AMP-treated cell lines We examined exponentially growing and cyclic AMP-treated cells by Northern blot analysis to confirm
DISCUSSION These experiments show a strikingly consistent pattern of cell cycle variation in messenger RNA concentration for the M2 subunit of ribonucleotide reductase. This cell cycle variation includes low levels in early G1 phase, and a very brief increase in M2 messenger RNA concentration at the GUS interphase, low levels in early to mid S phase, and a rise in later S phase and G2/M. The same basic cell cycle variation is present in hydroxyurea-resistant cells that are amplified in M2 activity, at least in part due to gene amplification (data not shown). The wild-type pattern of cell cycle variation in M2-specific messenger RNA concentration is also exhibited by cyclic AMP-dependent protein kinase-deficient cells, and by hydroxyurea-resistant cell lines both with and without cyclic AMP-dependent protein kinase activity. Thus, under normal circumstances neither PKA activity nor M2 gene amplification alters the pattern of cell cycle regulation of M2-specific message. Furthermore, M2 messenger RNA concentrations do not correlate with enzyme activity. Enzyme activity does correlate, however, with M2 protein concentration (Engstrom et al., 1985; Eriksson et al., 1984). Thus, in addition to transcriptional and post-translational regulation, it is possible that post-transcriptional regulation of ribonucleotide reductase plays a role in gene expression. This has been suggested for another DNA synthetic enzyme, thymidine kinase (Sherley and Kelly, 19881, where efficiency of translation is thought to affect gene expression. The effect of cyclic AMP on M2-specific messenger RNA concentration is dramatically different in those cells possessing cyclic AMP-dependent protein kinase activity than those that are deficient. Wild-type cells are arrested in G1 phase by cyclic AMP and cyclic AMP analogs, a s shown in Figures 2 and 3. This arrest is accompanied by a diminution in messenger RNA concentration. In cyclic AMP-dependent protein kinasedeficient cells, however, there is no effect of cyclic AMP on messenger RNA concentrations, either in exponentially growing cells that remain exponentially growing after cyclic AMP treatment, or in those cells exposed to cyclic AMP and then elutriated into cell cycle-specific fractions. The progressive decline in M2 messenger RNA concentrations in wild-type cells exposed to cyclic AMP parallels their arrest in G1 and subsequent release from G1 arrest when cyclic AMP is washed out of the cells. However, i t appears that the messenger RNA concentration lags slightly behind the initial release
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EFFECT OF CYCLIC AMP
Fig. 3. Wild type S49 cells exposed to cyclic AMP reversibly arrest in GI with low M2-specific messenger RNA concentrations. Exponentially growing wild-type cells (exp) were exposed for 1, 2.4, 6, 12, 18, and 24 hours to 1 mM dibutyryl cyclic AMP. The ribonucleotide reductase activity (CDP reductase activity in percent of exponential cell
--Wild Type
Exp
CAMP
HYU Resistant HYU Resistant KIN* KINE X ~ CAMP
E X ~ CAMP
2.1 kb
Fig. 4. Northern blot analysis-10 kg total cellular RNA was electrophoresed through a formaldehyde agarose gel transferred t~ nylon filter paper then probed with the same cDNA that was used in previous experiments. The 2.1 kb RNA species was identified in wildtype, 500-new (HYU-resistant KIN') and 500-1-1 (HYU-resistant KIN-) cell lines and was increased in hydroxyurea-resistant cells that were amplified in CDP reductase activity. Only PKA-positive cells show diminution of this transcript when cells were exposed to cyclic AMP.
from G1 arrest and at least does not precede the release from G1 arrest. This suggests that there may not be a direct cause and effect relationship between the diminution in M2-specific messenger RNA concentrations and cell cycle arrest. Indeed, deoxyadenosine also induces cell cycle arrest in early G1 with low M2 specific mRNA concentrations (Albert et al., unpublished observations) probably through a different mechanism than cyclic AMP. The variation of messenger RNA concentration could come a s a result of either transcriptional regulation (Roesler et al., 1988) or changes in message stability
culture control), cell cycle distribution, and M2 message concentrations are shown in the left panel. The right panel shows the same data for cells exposed to cyclic AMP for 24 hours then washed out and assayed at 0, 1, 2, 4, 6, 12, 18, and 24 hours after washout. Each dot is shown in duplicate to demonstrate reproducibility.
(i.e., message half-life), which has been thought to decline after translation (Rosenthal et al., 1980; Brock and Shapiro, 1983). It is possible that either of these processes could result in variation of message concentration. Cyclic AMP treatment results in an accumulation of cells in G1 phase with a decline in M2-specific messenger RNA concentration and a progressive decline in ribonucleotide reductase enzyme activity, whereas cells beyond the GUS interphase proceed through the cell cycle. Thus, these data suggest that a protein phosphorylated by cyclic AMP-dependent protein kinase could act as a transcriptional regulator in S49 cells and could participate in the cell cycle arrest. This effect may be mediated by a cyclic AMP-responsive element as has been shown for cyclic AMP-induced activation of gene transcription (Montminy and Bilezikjian, 1987) but to date it has only been described as a positive effector element. The alternative hypothesis that cyclic AMP affects message stability is partially supported by the presence of a n AU-rich sequence in the 3' flanking region of messenger RNA that is thought to confer instability (Shaw and Kamen, 1986).Further experiments will be necessary to determine which of these possibilities is correct.
ACKNOWLEDGMENTS We thank L. Thelander for the gift of the plasmid containing the M2 cDNA and Michael Becker for assistance in performing the Northern blot analysis and critical review of this manuscript. Technical support was provided by J. Kuchibhotla. These studies were supported by a n Arthritis Investigator Award from the Arthritis Foundation and Public Health Service grant CA46607 from the National Cancer Institute. LITERATURE CITED Akerblom, L., Ehrenberg, A,, Graslund, H., Lankinen, H., Reichard, P., and Thelander, L. (1981) Overproduction of the free radical of ribonucleotide reductase in hydroxyurea-resistant mouse fibroblast 3T6 cells. Roc. Natl. Acad. Sci. U S A . , 78t2159-2163. Albert, D.A., and Gudas, L.J. (1985) Deoxyribonucleoside triphosphate metabolism during the cell cycle. J. Biol. Chem., 260:679684. Albert. D.A.. Gudas. L.J.. and Nodzenski. E. (1987)Deoxvribonucle-
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