Regulation of mammalian ribonucleotide reductase by the tumor promoters and protein phosphatase inhibitors okadaic acid and calyculin A ROBERTA. R. HURTAAND JIM A.
WRIGHT'
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Departments of Microbiology, Biochemistry and Molecular Biology, and the Manitoba Institute of Cell Biology, University of Manitoba, 100 OIivia Street, Winnipeg, Man., Canada R3E OV9 Received March 3, 1992 HURTA,R. A. R., and WRIGHT,J. A. 1992. Regulation of mammalian ribonucleotide reductase by the tumor promoters and protein phosphatase inhibitors okadaic acid and calyculin A. Biochem. Cell Biol. 70: 1081-1087. A rapid elevation of ribonucleotide reductase activity was observed with BALB c/3T3 fibroblasts treated with 10 nM okadaic acid, a nonphorbol ester tumor promoter and protein phosphatase inhibitor. Northern blot analysis of the two components of ribonucleotide reductase (R1 and R2) showed a marked elevation of R1 and R2 mRNA expression. Western blot analysis with R1 and R2 specific monoclonal antibodies indicated that the increase in ribonucleotide reductase activity was primarily due to the elevation of the R2 rather than the R1 protein during treatment with okadaic acid. The okadaic acid induced elevations in R1 and R2 message levels occurred without a detectable change in the proportion of cells in S phase and were blocked by treatment of cells with actinomycin D, indicating the importance of the reductase transcriptional process in responding to the action of okadaic acid. Furthermore, down-regulation of protein kinase C with 12-0-tetradecanoylphorbol-13-acetate pretreatment abrogated the okadaic acid mediated elevation of ribonucleotide reductase mRNAs, consistent with the involvement of this signal pathway in the regulation of ribonucleotide reductase and the effects of okadaic acid. Treatment of cells with 2.5 nM calyculin A, another non-phorbol ester tumor promoter and protein phosphatase inhibitor, resulted in a rapid elevation of both R1 and R2 mRNA levels within 10 min of treatment. This first demonstration that the non-phorbol ester tumor promoters and protein phosphatase inhibitors can cause rapid alterations in ribonucleotide reductase gene expression suggests that (i) ribonucleotide reductase, particularly the R2 component, plays a fundamental role in the critical early events involved in the process of tumor promotion, and (ii) illustrates a role for cellular protein phosphatases in the regulation of ribonucleotide reductase and, through this process, the regulation of DNA synthesis. Key words: ribonucleotide reductase, DNA synthesis, okadaic acid, calyculin A, tumor promoter, protein phosphatase. HURTA, R. A. R., et WRIGHT, J. A. 1992. Regulation of mammalian ribonucleotide reductase by the tumor promoters and protein phosphatase inhibitors okadaic acid and calyculin A. Biochem. Cell Biol. 70 : 1081-1087. Nous avons observe une tlhation rapide de l'activitt de la ribonucltotide rtductase dans les fibroblastes BALB c/3T3 traitb avec I'acide okadai'que 10 nM, un ester non phorbol qui provoque des tumeurs et inhibe les prot6me phosphatases. L'analyse par transfert Northern des deux constituants de la ribonucltotide rtductase (R1 et R2) montre une tlkation marqute de l'expression des mRNA de R1 et R2, L'analyse par transfert Western avec des anticorps monoclonaux sptcifiques de R1 et R2 dtmontre que durant le traitement avec l'acide okadaique, l'augmentation de l'activitt de la ribonucltotide rtductase est d'abord due a l'tltvation de R2 plutat que de R1. L'tlhation du taux des messages de R1 et de R2 induite par l'acide okadai'que suwient sans changement dtcelable dans la proportion des cellules en phase S et elle est bloqute par traitement des cellules avec l'actinomycine D; cela prouve l'importance du processus transcriptionnel de la rCductase en rtponse l'action de l'acide okaddique. De plus, la rtgulation ntgative de la prottine kinase C prttraitte avec le 12-0-tttradtcanoylphorbol-13-acttateabroge l'tltvation des mRNA de la ribonucltotide rtductase induite par l'acide okaddique; cela confirme I'implication de cette voie signal dans la rtgulation de la ribonucltotide rCductase et les effets de l'acide okadaique. Le traitement des cellules avec la calyculine A, 2,s nM, un autre ester non phorbol qui provoque des tumeurs et inhibe les prottine phosphatases, entrafne une tltvation rapide du taux des mRNA de R1 et de R2 en moins de 10 min. Cette premiere dtmonstration que des esters non phorbol inducteurs de tumeurs et inhibiteurs des prottine phosphatases puissent causer des alterations rapides de l'expression des genes de la ribonuclCotide rtductase suggere que ( i ) la ribonucltotide rtductase, particulitrement le constituant R2, joue un rale fondamental dans les premiers htnements critiques impliquts dans le processus de promotion des tumeurs et (ii) illustre un r61e des prottine phosphatases cellulaires dans la rtgulation de la ribonucltotide rtductase et, par ce processus, de la rtgulation de la synthbe du DNA. Mots clgs : ribonucltotide rtductase, synthbe du DNA, acide okadaique, calyculine A, inducteur de tumeur, prottine phosphatase. [Traduit par la rtdaction]
Introduction ~ a m m a l i a nribonucleotide reductase plays a key role in the synthesis of DNA because it is solely responsible for the de novo reduction of ribonucleoside diphosphates to their corresponding deoxyribonucleoside diphosphate forms, ABBREVIATIONS: a-MEM, alpha minimal essential medium; FBS, fetal bovine serum; PBS, phosphate-buffered saline; GAPDH, glycera~dehyde-3-phosphatedehydrogenase; SDS, sodium dodecyl sulfate; IgG, immunoglobulin G ;kb, kilobase(s); TPA, 12-0-tetradecanoylphorbol-13-acetate. ' ~ u t h o rto whom all correspondence should be addressed. Printed in Canada / lmprime au Canada
which are required for DNA synthesis (Wright 1989). Ribonucleotide reductase is a highly controlled enzyme activity and the mammalian enzyme consists of two dissimilar protein components, ~1 and ~ 2which , are noncoordinately (Hopper 19-72; Choy et al. 1988; Wright 1989; w;ight et bl. i990). weight The R1 component, which is a dimer 170 000, contains substrate and multiple effector binding sites important for the complex allosteric regulation of the enzyme (Wright 1989; Wright et al. 1990). The level of the R1 subunit appears to be nearly constant throughout the
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cell cycle of actively proliferating cells (Mann et al. 1988). The R2 component, which is a dimer of molecular weight 90 000, contains nonheme iron and a unique tyrosyl-free radical required for ribonucleotide reduction (Wright 1989; Wright et al. 1990; McClarty et al. 1990). There is an S phase correlated increase in R 2 protein resulting from its de novo synthesis (Eriksson et al. 1984). Notably, the activity of ribonucleotide reductase is cell cycle dependent with maximal activity during S phase (Lewis et al. 1978; Thelander et al. 1985) and appears to be controlled by the synthesis and breakdown of protein R2, the limiting component for enzyme activity (Wright et al. 1990). Okadaic acid, a polyether compound of a C38fatty acid, is a potent nonphorbol ester tumor promoter for animal cells (Suganuma et al. 1988). Unlike phorbol ester tumor promoters which activate protein kinase C, okadaic acid specifically inhibits the phosphoserine/phosphothreonine protein phosphatases 1 and 2A. No effect on other phosphatases or a variety of protein kinases has been observed (Haystead et al. 1989). When okadaic acid is added to intact cells, it causes an increase in the apparent phosphorylation of certain cellular proteins and modifies metabolism in the manner expected of a specific protein phosphatase inhibitor. We have investigated the effects of okadaic acid, in the short term, on the expression of the ribonucleotide reductase genes, to determine if there is a role for phosphoprotein phosphatases in the signal pathways modulating gene expression. In an attempt to confirm observations obtained with okadaic acid, these studies were extended to include calyculin A (another potent tumor promoter) and phosphoserine/phosphothreonine protein phosphatase inhibitor (Suganuma et al. 1990). We show here for the first time that treatment with these inhibitors quickly and dramatically alters the expression of ribonucleotide reductase genes. Materials and methods Cell lines and culture conditions BALB c/3T3 mouse cell lines were routinely cultured at 37°C on plastic tissue culture plates (Lux Scientific, Ltd., Tucson, Ariz.) in a-MEM (Flow Laboratories) supplemented with antibiotics and 7% (v/v) FBS (Intergen, Co., Purchase, N.Y.). Logarithmically growing cells were treated with medium supplemented with okadaic acid (L.C. Services Corporation, Woburn, Mass.), dissolved in N,N-dimethylformamide. The final concentrations were 10 nM okadaic acid and 0.001% formamide; control plates received medium containing 0.001% formamide alone. The cells were incubated for various times with okadaic acid and then harvested (Hards and Wright 1981). Logarithmically growing cells were also treated with medium supplemented with calyculin A (L.C. Services Corporation) dissolved in absolute ethanol, for 10 min. The final concentrations of calyculin A used were 2.5, 5.0, and 10 nM, respectively; and control plates received medium containing 0.001 VO ethanol alone. Following the 10-min incubation, cells were harvested (Hards and Wright 1981). Ribonucleotide reductase assay Exponentially growing cells were plated at a density of approximately 3 x lo6 cells/l50-mm plastic tissue culture plates containing a-MEM + 7% FBS and incubated for 24 h at 37°C. Cells were then exposed to 10 nM okadaic acid dissolved in N,N-dimethylformamide for 1,4, and 8 h, respectively. Control plates received only the solvent. Following these manipulations, cells were removed, pelleted by centrifugation, washed with ice cold PBS twice, repelleted, and assayed directly or frozen at - 70°C until required. Enzyme preparations containing 2.5-4 mg protein/mL
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were used to assay for ribonucleotide reductase by the modified method of Steeper and Stuart (1970), using [ I 4 c ] c ~ (Moravek p Biochemicals, Inc., Brea, Calif.) as the substrate and Crotalus atrox snake venom, dissolved in 0.1 M Hepes (pH 8.0) containing 10 mM MgCl,, to hydrolyse the nucleotides to nucleosides as previously described (Lewis et al. 1978; Choy et al. 1988; Hurta and Wright 1990a). The reaction mixture contained in a final volume of 150 pL the following: [ 1 4 c ] c ~ (0.05 p &i (1 Ci = 37 GBq). 7.5 nmol), dithiothreitol (900 nmol), magnesium acetate (600 nmol), ATP (300 nmol), and an appropriate quantity of enzyme preparation. Reactions were initiated by the addition of enzyme and then carried out for 20 min at 37OC for CDP reduction. The reaction was terminated by boiling for 5 min. The nucleotides were converted to nucleosides by treatment for 1 h at 37OC with 2 mg/assay of Crotalus atrox venom. The reaction again was terminated by boiling for 5 min and then 0.5 mL of distilled water was added to each assay tube. The heat-precipitable material was removed by centrifugation. The supernatant was retained and the deoxycytidine was separated from the cytidine compounds on 5 x 80 mm Dowex-1-borate columns (Bio-Rad Laboratories). Briefly, the separation depended on the formation of a complex between ribonucleosides and borate ions in the column resin. Deoxyribonucleosides do not possess a cis-diol and hence cannot form a complex. Deoxycytidine was eluted from the column with 5 mL of distilled water. Ten millilitres of Scintiverse (Fisher Scientific) was then added. Radioactivity was determined in a Beckman model LS7800 liquid scintillation spectrophotometer. Northern blot analysis A rapid RNA extraction method was used to prepare total cellular RNA (Gough 1988), which was subjected to electrophoresis through 1% formaldehyde - agarose gels, followed by transfer to nylon membranes (Nytran, Schleicher and Schuell, Keene, N.H.). Blots were prehybridized and hybridized as described previously (Wright et al. 1987; McClarty et al. 1987, 1990). Hybridization occurred in the presence of either a 3Z~-labelled NcoI-generated fragment containing the cDNA of clone 65 (Rl) or the PstIgenerated fragment of clone 10 (R2)(McClarty et al. 1987). Probes were labelled using an oligolabelling kit (Pharmacia LKB Biotechnology, Inc., Uppsala, Sweden) and (a-3z~)-labelled dCTP (Amersham, Oakville, Ont.). Blots were washed and autoradiography was performed as outlined previously (McClarty et al. 1990; Hurta et al. 1991). Loading of RNA was estimated by probing with a plasmid containing the GAPDH gene labelled by nick translation (Choy et al. 1989). Densitometric analysis of appropriate autoradiograms was carried out using a Beckman DU-8 gel scanning spectrophotometer and the one-dimensional Bio-Rad program (Bio-Rad) (Hurta and Wright 1990a, 1990b). Western blot analysis Procedures used were as described previously (McClarty et al. 1987; Choy et al. 1988; Hurta and Wright 1990b). Following cell extract preparations, total protein content was determined and an aliquot was analyzed on 10% linear SDS-polyacrylarnide gel (Choy et al. 1988). After protein transfer and blocking, membranes were incubated either with JB4 anti-R2 monoclonal antibody (McClarty et al. 1987; Choy et al. 1988) or with AD203 anti-R1 monoclonal antibody (McClarty et al. 1987; Choy et al. 1988). Alkaline phosphatase conjugated either to rabbit anti-rat IgG (Sigma) or rabbit anti-mouse IgG (Sigma) was used for protein subunit R2 or subunit R1 detection, respectively. Assay for DNA synthesis DNA synthesis in cells cultured in six-well Nunclon plates (Nunc, Roskilde, Denmark) was measured by incorporation of [3~]thymidine (ICN Radiochemicals, Costa Mesa, Calif.) for 2 h. Growth medium was then removed and cells were detached with 0.3% buffered trypsin solution added to the wells for 30 min at 37°C. Cellular material was then processed further as previously
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1. Elevation of ribonucleotide reductase activity by okadaic TABLE acid treatment
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Hours of 10 nM okadaic acid treatment
Ribonucleotide reductase activity* Fold (nM CDP reduced/(h.mg protein)) increase
NOTE:The ribonucleotide reductase activity of control cells treated with N,N-dimethylformamidealone (0.001%) for up to 8 h showed no change in ribonucleotide reductase enzyme activity. Ribonucleotide reductase activity (two independent measurements done in duplicate) was 0.94 ? 0.03 nM CDP reduced/(h.mg protein) and 0.90 0.02 nM CDP reduced/(h.mg protein) for control cells (no formamide present) and cells treated with 0.001% formamide for 8 h, respectively. Similar results were found at 1 and 4 h exposure of cells to 0.001% formamide alone. *Values shown are the average + SE of three independent determinations of ribonucleotide reductase activity.
*
described (Hurta et al. 1991; Hurta and Wright 1992). Radioactivity was determined by liquid scintillation spectroscopy using a model L57800 scintillation counter (Beckman, Mississauga, Ont.). Results Effect of okadaic acid on the level of ribonucleotide reductase activity Proliferating BALB c/3T3 fibroblasts were treated with 10 nM okadaic acid. An elevation of ribonucleotide reductase activity by nearly fourfold was observed with 1 h of treatment of cells with the tumor promoter (Table 1). Elevation in enzyme activity was maintained during the course of the study, up to 8 h (Table 1). In control experiments, where cells were exposed to 0.001% formamide alone (the vehicle within which okadaic acid was dissolved) for 1, 4, and 8 h, respectively, no elevation of ribonucleotide reductase activity was observed (Table 1). DNA synthesis in the presence or absence of okadaic acid Since ribonucleotide reductase is markedly elevated in mammalian cells during DNA synthesis (Wright 1989; Wright et al. 1990), we examined the possibility that treatment of BALB c/3T3 cells with okadaic acid may shift a significant proportion of the cell population into S phase, which could be responsible for the increased enzyme activity. This point was investigated directly by estimating the incorporation of [3~]thymidineinto DNA at several time periods over an 8-h exposure of BALB c/3T3 cells to okadaic acid during which increased ribonucleotide reductase activity was observed (Table 1). Figure 1 shows that there were no significant differences in DNA synthesis rates, between cells treated with okadaic acid for up to 8 h and cells grown in the absence of okadaic acid. Therefore, the elevation in ribonucleotide reductase activity occurs in the absence of any detectable changes in the rates of DNA synthesis and this elevation is not due to an unusual shift of the cell population into S phase.
Effect of okadaic acid on R l and R2 mRNA levels To determine if the drug-induced increase in ribonucleotide reductase activity was accompanied by elevations in message levels for the two components of ribonucleotide
0
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4 8 TIME (h) FIG. 1. [3~]~hymidine incorporation into DNA as a measure of DNA synthesis during early induction of ribonucleotide reductase activity. BALB c/3T3 cells (104/well) were cultured in the absence ( - ) or presence ( + ) of okadaic acid (10 nM) for the times indicated. NA indicates no additions, whereas ( - ) indicates the presence of 0.001% formamide. Cells were then pulsed with [3~]thymidine for 2 h and the incorporation of label into trichloroacetic acid precipitable material was determined. Data shown are from two independent experiments done in triplicate.
reductase, BALB c/3T3 cells were cultured in the presence of 10 nM okadaic acid for various times, and the levels of R1 and R2 mRNAs were determined by Northern blots of 10 kg RNA/sample. Results of Northern blot analysis using R 1-specific cDNA as hybridization probe revealed that the R1 message was elevated approximately fourfold after 1 h treatment with okadaic acid, and this elevation was sustained following 4 and 8 h treatment with okadaic acid, respectively (Fig. 2A). R2-specific cDNA probed blots showed a dramatic elevation of both R2 transcripts (2.1 and 1.6 kb) usually observed in mouse cells (Wright et al. 1987; McClarty et al. 1987). Approximately seven- to eight-fold increases in R2 mRNA expression were observed at 1,4, and 8 h treatment with okadaic acid, respectively (Fig. 2A). As a loading control for each lane, the Northern blots were stripped and reprobed with GAPDH-specificcDNA (Edwards et al. 1985) (Fig. 2A). No change in R1 and R2 message levels was found in control cells treated with the solvent (N,N-dimethylformamide) used to dissolve okadaic acid and incubated for 8 h (data not shown). Okadaic acid (10 nM) treatment of BALB c/3T3 cells also resulted in an induction of junB mRNA transcripts. This gene is a member of the jun family of immediate early genes (Pertovaara et al. 1989). Northern blot analysis revealed 4.6-, 5.5, and 4.5-fold increases in junB mRNA expression at 1, 4, and 8 h of treatment of cells with okadaic acid (data not shown). These observations are consistent with other results that show alterations in expression of mRNA transcripts of members of the jun family (and of the fos family) by okadaic acid (Thevenin et al. 1991), and indicate that the tumor promoter is functioning in our studies as expected, based upon previous work.
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GAPDH FIG. 2. (A) Elevation of R2 and R1 message levels at various times in the presence of 10 nM okadaic acid. Northern blot analysis of R2, R1, and GAPDH mRNA levels in BALB c/3T3 cells cultured in the absence (0) or presence of okadaic acid for 1, 4, and 8 h, respectively. The R2, R1, and GAPDH autoradiograms were exposed for 24, 72, and 48 h, respectively, at -70°C with intensifying screens. (B) Rapid elevation of R2 and R1 message levels in the presence of 10 nM okadaic acid. Northern blot analysis of R2, R1, and GAPDH mRNA levels in BALB c/3T3 cells cultured in the absence (0) or presence of okadaic acid for 5 min. R2, R1, and GAPDH autoradiograms were exposed for 48, 72, and 24 h, respectively, at -70°C with intensifying screens.
Effect of calyculin A, another protein phosphatase inhibitor, on Rl and R2 mRNA levels Since okadaic acid treatment of BALB c/3T3 cells resulted in elevations of R1 and R2 mRNA expression, we questioned whether this response was due to protein phosphatase inhibition in general or whether this was an okadaic acid specific response. To address this question, we examined the effect of treatment of BALB c/3T3 cells with calyculin A, a protein phosphatase inhibitor. Calyculin A, like okadaic acid, is a potent inhibitor of phosphoserine/phosphothreonine protein phosphatases 1 and 2A and is a tumor promoter (Biolajan and Takai 1988; Suganuma et al. 1990). Although both compounds inhibit protein serinekhreonine phosphatases of the 2A class with very high potency, calyculin A is some 20- to 300-fold more Dotent than okadaic acid in inhibiting various forms ofathe class 1 phosphatases (Biolajan and Takai 1988). Like okadaic acid, calyculin A was able to induce R1 and R2 mRNA gene expression as shown in Fig. 3. R1 mRNA was found to be elevated approximately 2.9, 3.2, and 3.0-fold following an exposure of cells to 2.5,5.0, and 10 nM calyculin A, respectively, for 10 min. Similarly, R2 mRNA was elevated 4.3, 4.2, and 4.3-fold following treatment of cells with 2.5 nM, 5.0 nM, and 10 nM calyculin A, respectively, for 10 min. Since calyculin A was able to induce alterations in ribonucleotide reductase gene expression as early as 10 min, we investigated the possibility that okadaic acid might alter ribonucleotide reductase gene expression at earlier times than previously shown (< 1 h). Figure 2B shows that following
GAPDH FIG. 3. Elevation of R2 and R1 message levels at various concentrations of calyculin A. Northern blots of R2 and R1 mRNA levels in BALB c/3T3 cells cultured in the absence (a) or presence of 2.5 (b), 5.0 (c), or 10.0 nM (d) calyculin A, respectively. Loading controls were performed with GAPDH; autoradiograms were exposed for 48,24, and 48 h, respectively, at - 70°C with intensifying screens.
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okadaic acid treatment (10 nM), an elevation of R2 and R1 mRNA levels was observed as early as 5 min post-okadaic acid treatment. Increases of 4.4- and 5.1-fold in R2 and R1 mRNA levels, respectively, were noted. These observations, coupled with the finding that no significant changes in the rate of DNA synthesis were found when cells were treated with okadaic acid up to 8 h (Fig. I), strongly support the idea that the observed alterations in ribonucleotide reductase gene expression in the presence of the tumor promoters were not due to an unusual shift of the cell population into the S phase.
Effect of okadaic acid on R l and R2 protein levels To determine if the increases in ribonucleotide reductase message levels resulted in elevations in protein levels, BALB c/3T3 cells were cultured in the presence of 10 nM okadaic acid for 1, 4, and 8 h. Protein levels were determined by Western blot analysis. Although the R1-probed Northern blot shown in Fig. 2A indicated a fourfold increase in R1 mRNA with okadaic acid treated cells, a Western blot probed with monoclonal antibody specific for protein R1 showed no obvious detectable elevation in the levels of the protein in okadaic acid treated cells (Fig. 4). This is in sharp contrast to Western blots probed with R2-specific monoclonal antibody, in which a clear increase in R2 protein was detected (Fig. 4). A 3.5-fold increase in R2 protein levels at 1,4, and 8 h of okadaic acid treatment was noted (Fig. 4), and these changes in R2 protein resembled the observations
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HURTA AND WRIGHT
FIG. 4. Effect of okadaic acid (10 nM) on cellular R2 and R1 protein levels. (A) The R2 Western blot contained 100 pg protein for each lane, and was probed with a monoclonal antibody to the R2 protein and developed by an alkaline phosphatase linked second antibody. (B) The R1 Western blot contained 200 pg protein loaded for each lane, and was probed with a monoclonal antibody to the R1 protein and developed by an alkaline phosphatase linked second antibody. of R2 mRNA levels in okadaic acid treated cells (Fig. 2A). This shows that an elevation in R2 protein alone resulted in increased enzyme activity (Table I), although both proteins are required for ribonucleotide reduction (Wright 1989), and supports previous findings that the R2 protein is the limiting component for ribonucleotide reductase activity in normal proliferating mammalian cells (Wright et al. 1990).
Effect of okadaic acid treatment on transcription of Rl and R2 genes The possibility that the increases in R1 and R2 message levels observed following exposure to okadaic acid were due to changes in gene transcription rates was tested by pretreating BALB c/3T3 cells with the transcription blocker actinomycin D (Phillips and Crowthers 1986), prior to exposure of the cells to okadaic acid. As shown in Fig. 5, actinomycin D (5 pg/mL) prevented the increase in R1 and R2 gene expressions previously observed following exposure to okadaic acid, suggesting that at least in part, okadaic acid increases ribonucleotide reductase gene expression by altering the transcriptional process. Effect of protein kinase C down-regulation on okadaic acid dependent RI and R2 mRNA induction Okadaic acid is a non-phorbol ester type tumor promoter (Suganuma et al. 1988) and it has no effect on any of the
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FIG. 5. Actinomycin D prevents the okadaic acid induced elevation of R2 and R1 message levels. (A) Northern blots of R2 mRNA levels in the absence of actinomycin D and okadaic acid (a); in the absence of actinornycin D, but with 10 nM okadaic acid for 1 (b), 4 (c), and 8 h (d); in the presence of actinornycin D (5 pg/mL) without okadaic acid (e); and in the presence of actinomycin D (5 pg/mL) with okadaic acid (10 nM) for 1 (A, 4 (g), and 8 h (h). (B) Northern blots of R1 mRNA levels as described above. (C) GAPDH loading controls of R2 and R1 blots. R2, R1, and GAPDH autoradiograms were exposed for 48, 96, and 24 h, respectively. protein kinases tested to date, including protein kinase C (Erdodi et al. 1988; Haystead et al. 1989). However, inhibition of protein phosphatases allows the unopposed activity of protein kinases constitutively present in the cell and leads to enhanced phosphorylation of many of the substrates of ~ r o t e i nkinases (Sassa et al. 1989). Previous observations from our laboratory (Choy et al. -1989) showed that there was a transient increase in R2 gene expression following treatment of BALB c/3T3 cells with the tumor promoter TPA. The mechanism of action of TPA involves modification of protein kinase C activity (Nishizuka 1986). Therefore, we were interested in evaluating the possibility that okadaic acid may be inducing alterations in ribonucleotide reductase gene expression through a mechanism that involves protein kinase C. To test this possibility, BALB c/3T3 cells were treated with 0.1 pM TPA for 48 h to downregulate protein kinase C activity (Young et al. 1987). Previous studies have indicated that R1 and R2 gene expressions are at approximately untreated control levels after 24-48 h of exposure to 0.1 pM TPA (Choy et al. 1989). Figure 6 clearly demonstrates that pretreatment of BALB c/3T3 cells with TPA for 48 h prevents the elevation of R1 and R2 message levels that are normally observed in the presence of okadaic acid. This result suggests a possible role for protein kinase C in the okadaic acid induced modulation of ribonucleotide reductase gene expression in BALB c/3T3 cells. Discussion Okadaic acid and calyculin A are potent inhibitors of protein phosphatase 1 and phosphatase 2A, two of the four major protein phosphatases in the cytosol of mammalian
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FIG. 6. TPA prevents the okadaic acid induced elevation of R2 and R1 message levels. BALB c/3T3 cells were treated with 0.1 pM TPA prior to exposure to 10 nM okadaic acid for various times. Northern blots are shown for R2 (A) and R1 (B) mRNA levels in the absence of okadaic acid but with 0.001% formamide (a); the presence of okadaic acid for 1 (b), 4 (c), and 8 h (d), respectively; in the presence of 0.1 pM TPA (48 h) and okadaic acid (1 h) (e); with TPA and okadaic acid (4 h) (A;and with TPA and okadaic acid (8 h) (g). R1 and R2 mRNA levels from cells treated with 0.1 pM TPA alone (48 h) are shown in (h). (C) GAPDH loading controls for the R2 and R1 Northern blots are also shown. Autoradiograms were exposed for 24-72 h at - 70°C with intensifying screens. The left-hand side is also shown in Fig. 2A; it is included here for ease of comparison.
cells that dephosphorylate serine and threonine residues (Cohen et al. 1990; Suganuma et al. 1990). It has been shown that okadaic acid does not inhibit protein tyrosine phosphatases or kinases that have been tested (Haystead et al. 1989). It appears that okadaic acid and calyculin A exert their effects by causing a net increase in the prevailing levels of phosphorylated proteins, an effect which is equivalent in many ways to activating protein kinases including protein kinase C. Results from this study indicate for the first time that treatment of mammalian cells with a protein phosphatase inhibitor can cause a significant increase in ribonucleotide reductase activity, R1 and R2 message, and R2 protein. It should be noted that ribonucleotide reductase is a highly regulated, rate-limiting step in DNA synthesis (Wright 1989; Wright et al. 1990), whose activity is normally coupled to the S phase of the cell cycle (Lewis et al. 1978; Thelander et al. 1985; Wright et al. 1990). The okadaic acid and calyculin A induced alterations in ribonucleotide reductase observed in this study occurred quickly (within 5 and 10 min, respectively), suggesting that a block at S phase or an unusual movement of cells into S phase is not responsible for the demonstrated modifications in ribonucleotide reductase activity and gene expression. These results are in keeping with DNA synthesis studies, which showed that there was no change in DNA synthesis rates in the presence of okadaic acid for even as long as 8 h, and with several recent investigations from our laboratory showing that mammalian ribonucleotide reductase gene expression can be elevated in the absence of detectable changes in DNA synthesis (Choy et al. 1989; Hurta
from the point of view of gaining a better understanding of DNA synthesis regulation, to determine more precisely the mechanism(s) responsible for this novel control of ribonucleotide reductase. Interestingly, the okadaic acid induced elevations in R1 and R2 message levels were blocked by treatment of cells with actinomycin D, indicating an involvement of the reductase transcriptional process. Previous studies in our laboratory (Choy et al. 1989) have demonstrated that ribonucleotide reductase activity and gene expression can be transiently modulated by the phorbol ester tumor promoter TPA, which activates protein kinase C (Nishizuka 1986). The pathways responsible for connecting the signal generated by protein kinase C activation by TPA and the alterations in ribonucleotide reductase gene expression are not known. Interestingly, down-regulation of protein kinase C with 0.1 pM TPA pretreatment abrogated the okadaic acid mediated elevation of ribonucleotide reductase mRNAs, consistent with the involvement of this signal pathway in the regulation of ribonucleotide reductase and the effects of okadaic acid. In agreement with this idea is the finding that TPA response elements are recognized, for example by the APl (jun/fos) transcription factor (Angel et al. 1987), and that protein phosphatases 1 and 2A appear to be involved as negative regulators of AP1 in human Jurkat T cells (Thevinin et al. 1991). Our observation that okadaic acid treatment of BALB c/3T3 fibroblasts lead to an elevation in message levels of a member of the jun family is also in keeping with this view. However, the specifics of the intracellular signalling pathway(s) responsible for okadaic acid and calyculin A modulation of ribonucleotide reductase gene expression and the role of protein phosphatases in this process remains to be further elucidated. Such investigations are in progress. In summary, this first demonstration that non-phorbol ester tumor promoters and protein phosphatase inhibitors can cause alterations in ribonucleotide reductase gene expression suggests that (i) ribonucleotide reductase, in particular the R2 component, participates in the critical early events involved in the process of tumor promotion, and (ii) illustrates a role for cellular protein phosphatases in the regulation of ribonucleotide reductase and, through this process, the regulation of DNA synthesis.
Acknowledgements Financial support to J.A.W. for this study was provided by the National Cancer Institute of Canada (NCIC), and the Natural Sciences and Engineering Research Council of Canada. J.A.W. is a Terry Fox Senior Research Scientist of the NCIC. R.A.R.H. is a previous recipient of a postdoctoral stipend from the Sellers Foundation, University of Manitoba. Angel, P., Imagawa, M., Chiu, R., Stein, B., Imbra, R.J., Rahmsdorf, H.J., Jonat, C., Herrlich, P., and Karin, M. 1987. Phorbol ester-inducible genes contain a common cis element recognized by a TPA-modulated trans-acting factor. Cell, 49: 729-739. Biolajan, C., and Takai, A. 1988. Inhibitory effect of a marinesponge toxin, okadaic acid, on protein phosphatases. Biochem. J. 256: 283-290. Choy, B.K., McClarty, G.A., Chan, A.K., Thelander, L., and Wright, J.A. 1988. Molecular mechanisms of drug resistance
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involving ribonucleotide reductase: hydroxyurea resistance in a series of clonally related mouse cell lines selected in the presence of increasing drug concentrations. Cancer Res. 48: 2929-2035. Choy, B.K., McClarty, G.A., and Wright, J.A. 1989. Transient elevation of ribonucleotide reductase activity, M2 mRNA and M2 protein in BALB c/3T3 fibroblasts in the presence of 12-0tetradecanoylphorbol-13-acetate. Biochem. Biophys. Res. Commun. 162: 1417-1424. Cohen, P., Holmes, C.F.B., and Tsukitani, Y. 1990. Okadaic acid: a new probe for the study of cellular regulation. Trends Biochem. Sci. 15: 98-102. Edwards, D.R., Parfett, C.L.J., and Denhardt, D.T. 1985. Transcriptional regulation of two serum-induced RNAs in mouse fibroblasts: equivalence of one species to B2 repetitive elements. Mol. Cell. Biol. 5: 3280-3288. Erdodi, F., Rokolya, A., Di Salvo, J., Barany, M., and Barany, K. 1988. Effect of okadaic acid on phosphorylationdephosphorylation of myosin light chain in aortic smooth muscle homogenate. Biochem. Biophys. Res. Commun. 153: 156-161. 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: 11 695 - 11 700. Gough, N.M. 1988. Rapid and quantitative preparation of cytoplasmic RNA from small numbers of cells. Anal. Biochem. 173: 93-95. Hards, R.G., and Wright, J.A. 1981. N-carbamoyloxyurearesistant Chinese hamster ovary cells with elevated levels of ribonucleotide reductase activity. J. Cell. Physiol. 106: 309-319. Haystead, T.A.J., Sim, A.T.R., Carling, D., Honnor, R.C., Tsukitani, Y., Cohen, P., and Hardie, D.G. 1989. Effects of the tumor promoter okadaic acid on intracellular protein phosphorylation and metabolism. Nature (London), 337: 78-81. Hopper. S. 1972. Ribonucleotide reductase of rabbit bone marrow. I. Purification, properties, and separation into protein fractions. J. Biol. Chem. 247: 3336-3340. Hurta, R.A.R., and Wright, J.A. 1990~.Amplification of the genes for both components of ribonucleotide reductase in hydroxyurea resistant mammalian cells. Biochem. Biophys. Res. Commun. 167: 258-264. Hurta. R.A.R., and Wright, J.A. 1990b. Mammalian drug resistant mutants with multiple gene amplifications: genes encoding the M1 component of ribonucleotide reductase, the M2 component of ribonucleotide reductase, ornithine decarboxylase, p5-8, the H-subunit of ferritin and the L-subunit of ferritin. Biochim. Biophys. Acta, 1087: 165-172. Hurta, R.A.R., and Wright, J.A. 1992. Alterations in the activity and regulation of mammalian ribonucleotide reductase by chlorambucil, a DNA damaging agent. J. Biol. Chem. 267: 7066-7071. Hurta, R.A.R., Samuel, S.K., Greenberg, A.H., and Wright, J.A. 1991. Early induction of ribonucleotide reductase gene expression by transforming growth factor+, in malignant H-ras transformed cell lines. J. Biol. Chem. 266: 24 097 - 24 100. Lewis, W.H., Kuzik, B.A., and Wright, J.A. 1978. Assay of ribonucleotide reduction in nucleotide-permeable hamster cells. J. Cell. Physiol. 94: 287-298. Mann, G.J., Musgrove, E.A., Fox, R.M., and Thelander, L. 1988.
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Ribonucleotide reductase M1 subunit in cellular proliferation, quiescence, and differentiation. Cancer Res. 48: 5 151-5 156. McClarty, G.A., Chan, A.K., Choy, B.K., Engstrom, Y., Wright, J.A., and Thelander, L. 1987. Elevated expression of M1 and M2 components and drug-induced posttranscriptional modulation of ribonucleotide reductase in a hydroxyurea-resistantmouse cell line. Biochemistry, 26: 8004-801 1. McClarty, G.A., Chan, A.K., Choy, B.K., and Wright, J.A. 1990. Increased ferritin gene expression and the establishment of hydroxyurea resistance in mammalian cells. J. Biol. Chem. 265: 7539-7547. Nishizuka, Y. 1986. Studies and perspectives of protein kinase C. Science (Washington, D.C.), 233: 305-312. Pertovaara, L.. Sistonen, L., Timothy, J., Boss, T.J., Vogt, P.K., Keski-Oja, J., and Alitalo, L. 1989. Enhanced jun expression is an early genomic response to transforming growth factor /3 stimulation. Mol. Cell. Biol. 9: 1255-1262. Phillips, D.R., and Crowthers, D.M. 1986. Kinetics and specificity of drug-DNA interactions: an in vitro transcription assay. Biochemistry, 25: 7355-7362. Sassa, T., Richter, W.W., Uda, N., Suganuma, M., Suguri, H., Yoshizuwa, S., Hirota, M., and Fujiki, H. 1989. Apparent activation of protein kinases by okadaic acid class tumor promoters. Biochem. Biophys. Res. Commun. 159: 939-944. Steeper, J.R., and Stuart, C.D. 1970. A rapid assay for CDP reductase activity in mammalian cell extracts. Anal. Biochem. 34: 123-130. Suganuma, M., Fujiki, H., Suguri, H., Yoshizawa, S., Hirota, M., Nakayasu, M., Ojika, M., Wakamatsu, K., Yamada. K., and Sugimura, T. 1988. Okadaic acid: an additional nonphorbol-12-tetradecanoate-13-acetate type tumor promoter. Proc. Natl. Acad. Sci. U.S.A. 85: 1768-1771. Suganuma, M., Fujiki. H., Furuya-Suguri, H., Yoshizawa, S., Yasumoto, S., Kato, Y., Fusetani, N., and Sugimura, T. 1990. Calyculin A, an inhibitor of protein phosphatases, a potent tumor promoter on SD-1 mouse skin. Cancer Res. 50: 3521-3525. Thelander, M., Graslund, A., and Thelander, L. 1985. Subunit M2 of mammalian ribonucleotide reductase. Characterization of a homologous protein isolated from M2 overproducing mouse cells. J. Biol. Chem. 260: 2737-2741. Thevenin, C., Kim, S.-J., and Kehrl, J.H. 1991. Inhibition of protein phosphatases by okadaic acid induces APl in human T-cells. J. Biol. Chem. 266: 9363-9366. Wright, J.A. 1989. Altered mammalian ribonucleoside diphosphate reductase from mutant cell lines. Int. Encycl. Pharmacol. Ther. 128: 89-111. Wright, J.A., Alam, T.G., McClarty, G.A., Tagger, A.Y., and Thelander, L. 1987. Altered expression of ribonucleotide reductase and role of M2 gene amplification in hydroxyurea-resistant hamster, mouse, rat and human cell lines. Somatic Cell Mol. Genet. 13: 155-165. Wright, J.A., Chan, A.K., Choy, B.K., Hurta. R.A.R., McClarty, G.A., and Tagger, A.Y. 1990. Regulation and drug resistance mechanisms of mammalian ribonucleotide reductase and the significance to DNA synthesis. Biochem. Cell Biol. 68: 1364-1371. Young, S., Parker, P.J., Ullrich, A., and Stabel, S. 1987. Downregulation of protein kinase C is due to an increased rate of degradation. Biochem. J. 244: 775-779.
WRIGHT'
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Departments of Microbiology, Biochemistry and Molecular Biology, and the Manitoba Institute of Cell Biology, University of Manitoba, 100 OIivia Street, Winnipeg, Man., Canada R3E OV9 Received March 3, 1992 HURTA,R. A. R., and WRIGHT,J. A. 1992. Regulation of mammalian ribonucleotide reductase by the tumor promoters and protein phosphatase inhibitors okadaic acid and calyculin A. Biochem. Cell Biol. 70: 1081-1087. A rapid elevation of ribonucleotide reductase activity was observed with BALB c/3T3 fibroblasts treated with 10 nM okadaic acid, a nonphorbol ester tumor promoter and protein phosphatase inhibitor. Northern blot analysis of the two components of ribonucleotide reductase (R1 and R2) showed a marked elevation of R1 and R2 mRNA expression. Western blot analysis with R1 and R2 specific monoclonal antibodies indicated that the increase in ribonucleotide reductase activity was primarily due to the elevation of the R2 rather than the R1 protein during treatment with okadaic acid. The okadaic acid induced elevations in R1 and R2 message levels occurred without a detectable change in the proportion of cells in S phase and were blocked by treatment of cells with actinomycin D, indicating the importance of the reductase transcriptional process in responding to the action of okadaic acid. Furthermore, down-regulation of protein kinase C with 12-0-tetradecanoylphorbol-13-acetate pretreatment abrogated the okadaic acid mediated elevation of ribonucleotide reductase mRNAs, consistent with the involvement of this signal pathway in the regulation of ribonucleotide reductase and the effects of okadaic acid. Treatment of cells with 2.5 nM calyculin A, another non-phorbol ester tumor promoter and protein phosphatase inhibitor, resulted in a rapid elevation of both R1 and R2 mRNA levels within 10 min of treatment. This first demonstration that the non-phorbol ester tumor promoters and protein phosphatase inhibitors can cause rapid alterations in ribonucleotide reductase gene expression suggests that (i) ribonucleotide reductase, particularly the R2 component, plays a fundamental role in the critical early events involved in the process of tumor promotion, and (ii) illustrates a role for cellular protein phosphatases in the regulation of ribonucleotide reductase and, through this process, the regulation of DNA synthesis. Key words: ribonucleotide reductase, DNA synthesis, okadaic acid, calyculin A, tumor promoter, protein phosphatase. HURTA, R. A. R., et WRIGHT, J. A. 1992. Regulation of mammalian ribonucleotide reductase by the tumor promoters and protein phosphatase inhibitors okadaic acid and calyculin A. Biochem. Cell Biol. 70 : 1081-1087. Nous avons observe une tlhation rapide de l'activitt de la ribonucltotide rtductase dans les fibroblastes BALB c/3T3 traitb avec I'acide okadai'que 10 nM, un ester non phorbol qui provoque des tumeurs et inhibe les prot6me phosphatases. L'analyse par transfert Northern des deux constituants de la ribonucltotide rtductase (R1 et R2) montre une tlkation marqute de l'expression des mRNA de R1 et R2, L'analyse par transfert Western avec des anticorps monoclonaux sptcifiques de R1 et R2 dtmontre que durant le traitement avec l'acide okadaique, l'augmentation de l'activitt de la ribonucltotide rtductase est d'abord due a l'tltvation de R2 plutat que de R1. L'tlhation du taux des messages de R1 et de R2 induite par l'acide okadai'que suwient sans changement dtcelable dans la proportion des cellules en phase S et elle est bloqute par traitement des cellules avec l'actinomycine D; cela prouve l'importance du processus transcriptionnel de la rCductase en rtponse l'action de l'acide okaddique. De plus, la rtgulation ntgative de la prottine kinase C prttraitte avec le 12-0-tttradtcanoylphorbol-13-acttateabroge l'tltvation des mRNA de la ribonucltotide rtductase induite par l'acide okaddique; cela confirme I'implication de cette voie signal dans la rtgulation de la ribonucltotide rCductase et les effets de l'acide okadaique. Le traitement des cellules avec la calyculine A, 2,s nM, un autre ester non phorbol qui provoque des tumeurs et inhibe les prottine phosphatases, entrafne une tltvation rapide du taux des mRNA de R1 et de R2 en moins de 10 min. Cette premiere dtmonstration que des esters non phorbol inducteurs de tumeurs et inhibiteurs des prottine phosphatases puissent causer des alterations rapides de l'expression des genes de la ribonuclCotide rtductase suggere que ( i ) la ribonucltotide rtductase, particulitrement le constituant R2, joue un rale fondamental dans les premiers htnements critiques impliquts dans le processus de promotion des tumeurs et (ii) illustre un r61e des prottine phosphatases cellulaires dans la rtgulation de la ribonucltotide rtductase et, par ce processus, de la rtgulation de la synthbe du DNA. Mots clgs : ribonucltotide rtductase, synthbe du DNA, acide okadaique, calyculine A, inducteur de tumeur, prottine phosphatase. [Traduit par la rtdaction]
Introduction ~ a m m a l i a nribonucleotide reductase plays a key role in the synthesis of DNA because it is solely responsible for the de novo reduction of ribonucleoside diphosphates to their corresponding deoxyribonucleoside diphosphate forms, ABBREVIATIONS: a-MEM, alpha minimal essential medium; FBS, fetal bovine serum; PBS, phosphate-buffered saline; GAPDH, glycera~dehyde-3-phosphatedehydrogenase; SDS, sodium dodecyl sulfate; IgG, immunoglobulin G ;kb, kilobase(s); TPA, 12-0-tetradecanoylphorbol-13-acetate. ' ~ u t h o rto whom all correspondence should be addressed. Printed in Canada / lmprime au Canada
which are required for DNA synthesis (Wright 1989). Ribonucleotide reductase is a highly controlled enzyme activity and the mammalian enzyme consists of two dissimilar protein components, ~1 and ~ 2which , are noncoordinately (Hopper 19-72; Choy et al. 1988; Wright 1989; w;ight et bl. i990). weight The R1 component, which is a dimer 170 000, contains substrate and multiple effector binding sites important for the complex allosteric regulation of the enzyme (Wright 1989; Wright et al. 1990). The level of the R1 subunit appears to be nearly constant throughout the
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cell cycle of actively proliferating cells (Mann et al. 1988). The R2 component, which is a dimer of molecular weight 90 000, contains nonheme iron and a unique tyrosyl-free radical required for ribonucleotide reduction (Wright 1989; Wright et al. 1990; McClarty et al. 1990). There is an S phase correlated increase in R 2 protein resulting from its de novo synthesis (Eriksson et al. 1984). Notably, the activity of ribonucleotide reductase is cell cycle dependent with maximal activity during S phase (Lewis et al. 1978; Thelander et al. 1985) and appears to be controlled by the synthesis and breakdown of protein R2, the limiting component for enzyme activity (Wright et al. 1990). Okadaic acid, a polyether compound of a C38fatty acid, is a potent nonphorbol ester tumor promoter for animal cells (Suganuma et al. 1988). Unlike phorbol ester tumor promoters which activate protein kinase C, okadaic acid specifically inhibits the phosphoserine/phosphothreonine protein phosphatases 1 and 2A. No effect on other phosphatases or a variety of protein kinases has been observed (Haystead et al. 1989). When okadaic acid is added to intact cells, it causes an increase in the apparent phosphorylation of certain cellular proteins and modifies metabolism in the manner expected of a specific protein phosphatase inhibitor. We have investigated the effects of okadaic acid, in the short term, on the expression of the ribonucleotide reductase genes, to determine if there is a role for phosphoprotein phosphatases in the signal pathways modulating gene expression. In an attempt to confirm observations obtained with okadaic acid, these studies were extended to include calyculin A (another potent tumor promoter) and phosphoserine/phosphothreonine protein phosphatase inhibitor (Suganuma et al. 1990). We show here for the first time that treatment with these inhibitors quickly and dramatically alters the expression of ribonucleotide reductase genes. Materials and methods Cell lines and culture conditions BALB c/3T3 mouse cell lines were routinely cultured at 37°C on plastic tissue culture plates (Lux Scientific, Ltd., Tucson, Ariz.) in a-MEM (Flow Laboratories) supplemented with antibiotics and 7% (v/v) FBS (Intergen, Co., Purchase, N.Y.). Logarithmically growing cells were treated with medium supplemented with okadaic acid (L.C. Services Corporation, Woburn, Mass.), dissolved in N,N-dimethylformamide. The final concentrations were 10 nM okadaic acid and 0.001% formamide; control plates received medium containing 0.001% formamide alone. The cells were incubated for various times with okadaic acid and then harvested (Hards and Wright 1981). Logarithmically growing cells were also treated with medium supplemented with calyculin A (L.C. Services Corporation) dissolved in absolute ethanol, for 10 min. The final concentrations of calyculin A used were 2.5, 5.0, and 10 nM, respectively; and control plates received medium containing 0.001 VO ethanol alone. Following the 10-min incubation, cells were harvested (Hards and Wright 1981). Ribonucleotide reductase assay Exponentially growing cells were plated at a density of approximately 3 x lo6 cells/l50-mm plastic tissue culture plates containing a-MEM + 7% FBS and incubated for 24 h at 37°C. Cells were then exposed to 10 nM okadaic acid dissolved in N,N-dimethylformamide for 1,4, and 8 h, respectively. Control plates received only the solvent. Following these manipulations, cells were removed, pelleted by centrifugation, washed with ice cold PBS twice, repelleted, and assayed directly or frozen at - 70°C until required. Enzyme preparations containing 2.5-4 mg protein/mL
1992
were used to assay for ribonucleotide reductase by the modified method of Steeper and Stuart (1970), using [ I 4 c ] c ~ (Moravek p Biochemicals, Inc., Brea, Calif.) as the substrate and Crotalus atrox snake venom, dissolved in 0.1 M Hepes (pH 8.0) containing 10 mM MgCl,, to hydrolyse the nucleotides to nucleosides as previously described (Lewis et al. 1978; Choy et al. 1988; Hurta and Wright 1990a). The reaction mixture contained in a final volume of 150 pL the following: [ 1 4 c ] c ~ (0.05 p &i (1 Ci = 37 GBq). 7.5 nmol), dithiothreitol (900 nmol), magnesium acetate (600 nmol), ATP (300 nmol), and an appropriate quantity of enzyme preparation. Reactions were initiated by the addition of enzyme and then carried out for 20 min at 37OC for CDP reduction. The reaction was terminated by boiling for 5 min. The nucleotides were converted to nucleosides by treatment for 1 h at 37OC with 2 mg/assay of Crotalus atrox venom. The reaction again was terminated by boiling for 5 min and then 0.5 mL of distilled water was added to each assay tube. The heat-precipitable material was removed by centrifugation. The supernatant was retained and the deoxycytidine was separated from the cytidine compounds on 5 x 80 mm Dowex-1-borate columns (Bio-Rad Laboratories). Briefly, the separation depended on the formation of a complex between ribonucleosides and borate ions in the column resin. Deoxyribonucleosides do not possess a cis-diol and hence cannot form a complex. Deoxycytidine was eluted from the column with 5 mL of distilled water. Ten millilitres of Scintiverse (Fisher Scientific) was then added. Radioactivity was determined in a Beckman model LS7800 liquid scintillation spectrophotometer. Northern blot analysis A rapid RNA extraction method was used to prepare total cellular RNA (Gough 1988), which was subjected to electrophoresis through 1% formaldehyde - agarose gels, followed by transfer to nylon membranes (Nytran, Schleicher and Schuell, Keene, N.H.). Blots were prehybridized and hybridized as described previously (Wright et al. 1987; McClarty et al. 1987, 1990). Hybridization occurred in the presence of either a 3Z~-labelled NcoI-generated fragment containing the cDNA of clone 65 (Rl) or the PstIgenerated fragment of clone 10 (R2)(McClarty et al. 1987). Probes were labelled using an oligolabelling kit (Pharmacia LKB Biotechnology, Inc., Uppsala, Sweden) and (a-3z~)-labelled dCTP (Amersham, Oakville, Ont.). Blots were washed and autoradiography was performed as outlined previously (McClarty et al. 1990; Hurta et al. 1991). Loading of RNA was estimated by probing with a plasmid containing the GAPDH gene labelled by nick translation (Choy et al. 1989). Densitometric analysis of appropriate autoradiograms was carried out using a Beckman DU-8 gel scanning spectrophotometer and the one-dimensional Bio-Rad program (Bio-Rad) (Hurta and Wright 1990a, 1990b). Western blot analysis Procedures used were as described previously (McClarty et al. 1987; Choy et al. 1988; Hurta and Wright 1990b). Following cell extract preparations, total protein content was determined and an aliquot was analyzed on 10% linear SDS-polyacrylarnide gel (Choy et al. 1988). After protein transfer and blocking, membranes were incubated either with JB4 anti-R2 monoclonal antibody (McClarty et al. 1987; Choy et al. 1988) or with AD203 anti-R1 monoclonal antibody (McClarty et al. 1987; Choy et al. 1988). Alkaline phosphatase conjugated either to rabbit anti-rat IgG (Sigma) or rabbit anti-mouse IgG (Sigma) was used for protein subunit R2 or subunit R1 detection, respectively. Assay for DNA synthesis DNA synthesis in cells cultured in six-well Nunclon plates (Nunc, Roskilde, Denmark) was measured by incorporation of [3~]thymidine (ICN Radiochemicals, Costa Mesa, Calif.) for 2 h. Growth medium was then removed and cells were detached with 0.3% buffered trypsin solution added to the wells for 30 min at 37°C. Cellular material was then processed further as previously
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1. Elevation of ribonucleotide reductase activity by okadaic TABLE acid treatment
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Hours of 10 nM okadaic acid treatment
Ribonucleotide reductase activity* Fold (nM CDP reduced/(h.mg protein)) increase
NOTE:The ribonucleotide reductase activity of control cells treated with N,N-dimethylformamidealone (0.001%) for up to 8 h showed no change in ribonucleotide reductase enzyme activity. Ribonucleotide reductase activity (two independent measurements done in duplicate) was 0.94 ? 0.03 nM CDP reduced/(h.mg protein) and 0.90 0.02 nM CDP reduced/(h.mg protein) for control cells (no formamide present) and cells treated with 0.001% formamide for 8 h, respectively. Similar results were found at 1 and 4 h exposure of cells to 0.001% formamide alone. *Values shown are the average + SE of three independent determinations of ribonucleotide reductase activity.
*
described (Hurta et al. 1991; Hurta and Wright 1992). Radioactivity was determined by liquid scintillation spectroscopy using a model L57800 scintillation counter (Beckman, Mississauga, Ont.). Results Effect of okadaic acid on the level of ribonucleotide reductase activity Proliferating BALB c/3T3 fibroblasts were treated with 10 nM okadaic acid. An elevation of ribonucleotide reductase activity by nearly fourfold was observed with 1 h of treatment of cells with the tumor promoter (Table 1). Elevation in enzyme activity was maintained during the course of the study, up to 8 h (Table 1). In control experiments, where cells were exposed to 0.001% formamide alone (the vehicle within which okadaic acid was dissolved) for 1, 4, and 8 h, respectively, no elevation of ribonucleotide reductase activity was observed (Table 1). DNA synthesis in the presence or absence of okadaic acid Since ribonucleotide reductase is markedly elevated in mammalian cells during DNA synthesis (Wright 1989; Wright et al. 1990), we examined the possibility that treatment of BALB c/3T3 cells with okadaic acid may shift a significant proportion of the cell population into S phase, which could be responsible for the increased enzyme activity. This point was investigated directly by estimating the incorporation of [3~]thymidineinto DNA at several time periods over an 8-h exposure of BALB c/3T3 cells to okadaic acid during which increased ribonucleotide reductase activity was observed (Table 1). Figure 1 shows that there were no significant differences in DNA synthesis rates, between cells treated with okadaic acid for up to 8 h and cells grown in the absence of okadaic acid. Therefore, the elevation in ribonucleotide reductase activity occurs in the absence of any detectable changes in the rates of DNA synthesis and this elevation is not due to an unusual shift of the cell population into S phase.
Effect of okadaic acid on R l and R2 mRNA levels To determine if the drug-induced increase in ribonucleotide reductase activity was accompanied by elevations in message levels for the two components of ribonucleotide
0
1
4 8 TIME (h) FIG. 1. [3~]~hymidine incorporation into DNA as a measure of DNA synthesis during early induction of ribonucleotide reductase activity. BALB c/3T3 cells (104/well) were cultured in the absence ( - ) or presence ( + ) of okadaic acid (10 nM) for the times indicated. NA indicates no additions, whereas ( - ) indicates the presence of 0.001% formamide. Cells were then pulsed with [3~]thymidine for 2 h and the incorporation of label into trichloroacetic acid precipitable material was determined. Data shown are from two independent experiments done in triplicate.
reductase, BALB c/3T3 cells were cultured in the presence of 10 nM okadaic acid for various times, and the levels of R1 and R2 mRNAs were determined by Northern blots of 10 kg RNA/sample. Results of Northern blot analysis using R 1-specific cDNA as hybridization probe revealed that the R1 message was elevated approximately fourfold after 1 h treatment with okadaic acid, and this elevation was sustained following 4 and 8 h treatment with okadaic acid, respectively (Fig. 2A). R2-specific cDNA probed blots showed a dramatic elevation of both R2 transcripts (2.1 and 1.6 kb) usually observed in mouse cells (Wright et al. 1987; McClarty et al. 1987). Approximately seven- to eight-fold increases in R2 mRNA expression were observed at 1,4, and 8 h treatment with okadaic acid, respectively (Fig. 2A). As a loading control for each lane, the Northern blots were stripped and reprobed with GAPDH-specificcDNA (Edwards et al. 1985) (Fig. 2A). No change in R1 and R2 message levels was found in control cells treated with the solvent (N,N-dimethylformamide) used to dissolve okadaic acid and incubated for 8 h (data not shown). Okadaic acid (10 nM) treatment of BALB c/3T3 cells also resulted in an induction of junB mRNA transcripts. This gene is a member of the jun family of immediate early genes (Pertovaara et al. 1989). Northern blot analysis revealed 4.6-, 5.5, and 4.5-fold increases in junB mRNA expression at 1, 4, and 8 h of treatment of cells with okadaic acid (data not shown). These observations are consistent with other results that show alterations in expression of mRNA transcripts of members of the jun family (and of the fos family) by okadaic acid (Thevenin et al. 1991), and indicate that the tumor promoter is functioning in our studies as expected, based upon previous work.
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GAPDH FIG. 2. (A) Elevation of R2 and R1 message levels at various times in the presence of 10 nM okadaic acid. Northern blot analysis of R2, R1, and GAPDH mRNA levels in BALB c/3T3 cells cultured in the absence (0) or presence of okadaic acid for 1, 4, and 8 h, respectively. The R2, R1, and GAPDH autoradiograms were exposed for 24, 72, and 48 h, respectively, at -70°C with intensifying screens. (B) Rapid elevation of R2 and R1 message levels in the presence of 10 nM okadaic acid. Northern blot analysis of R2, R1, and GAPDH mRNA levels in BALB c/3T3 cells cultured in the absence (0) or presence of okadaic acid for 5 min. R2, R1, and GAPDH autoradiograms were exposed for 48, 72, and 24 h, respectively, at -70°C with intensifying screens.
Effect of calyculin A, another protein phosphatase inhibitor, on Rl and R2 mRNA levels Since okadaic acid treatment of BALB c/3T3 cells resulted in elevations of R1 and R2 mRNA expression, we questioned whether this response was due to protein phosphatase inhibition in general or whether this was an okadaic acid specific response. To address this question, we examined the effect of treatment of BALB c/3T3 cells with calyculin A, a protein phosphatase inhibitor. Calyculin A, like okadaic acid, is a potent inhibitor of phosphoserine/phosphothreonine protein phosphatases 1 and 2A and is a tumor promoter (Biolajan and Takai 1988; Suganuma et al. 1990). Although both compounds inhibit protein serinekhreonine phosphatases of the 2A class with very high potency, calyculin A is some 20- to 300-fold more Dotent than okadaic acid in inhibiting various forms ofathe class 1 phosphatases (Biolajan and Takai 1988). Like okadaic acid, calyculin A was able to induce R1 and R2 mRNA gene expression as shown in Fig. 3. R1 mRNA was found to be elevated approximately 2.9, 3.2, and 3.0-fold following an exposure of cells to 2.5,5.0, and 10 nM calyculin A, respectively, for 10 min. Similarly, R2 mRNA was elevated 4.3, 4.2, and 4.3-fold following treatment of cells with 2.5 nM, 5.0 nM, and 10 nM calyculin A, respectively, for 10 min. Since calyculin A was able to induce alterations in ribonucleotide reductase gene expression as early as 10 min, we investigated the possibility that okadaic acid might alter ribonucleotide reductase gene expression at earlier times than previously shown (< 1 h). Figure 2B shows that following
GAPDH FIG. 3. Elevation of R2 and R1 message levels at various concentrations of calyculin A. Northern blots of R2 and R1 mRNA levels in BALB c/3T3 cells cultured in the absence (a) or presence of 2.5 (b), 5.0 (c), or 10.0 nM (d) calyculin A, respectively. Loading controls were performed with GAPDH; autoradiograms were exposed for 48,24, and 48 h, respectively, at - 70°C with intensifying screens.
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okadaic acid treatment (10 nM), an elevation of R2 and R1 mRNA levels was observed as early as 5 min post-okadaic acid treatment. Increases of 4.4- and 5.1-fold in R2 and R1 mRNA levels, respectively, were noted. These observations, coupled with the finding that no significant changes in the rate of DNA synthesis were found when cells were treated with okadaic acid up to 8 h (Fig. I), strongly support the idea that the observed alterations in ribonucleotide reductase gene expression in the presence of the tumor promoters were not due to an unusual shift of the cell population into the S phase.
Effect of okadaic acid on R l and R2 protein levels To determine if the increases in ribonucleotide reductase message levels resulted in elevations in protein levels, BALB c/3T3 cells were cultured in the presence of 10 nM okadaic acid for 1, 4, and 8 h. Protein levels were determined by Western blot analysis. Although the R1-probed Northern blot shown in Fig. 2A indicated a fourfold increase in R1 mRNA with okadaic acid treated cells, a Western blot probed with monoclonal antibody specific for protein R1 showed no obvious detectable elevation in the levels of the protein in okadaic acid treated cells (Fig. 4). This is in sharp contrast to Western blots probed with R2-specific monoclonal antibody, in which a clear increase in R2 protein was detected (Fig. 4). A 3.5-fold increase in R2 protein levels at 1,4, and 8 h of okadaic acid treatment was noted (Fig. 4), and these changes in R2 protein resembled the observations
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FIG. 4. Effect of okadaic acid (10 nM) on cellular R2 and R1 protein levels. (A) The R2 Western blot contained 100 pg protein for each lane, and was probed with a monoclonal antibody to the R2 protein and developed by an alkaline phosphatase linked second antibody. (B) The R1 Western blot contained 200 pg protein loaded for each lane, and was probed with a monoclonal antibody to the R1 protein and developed by an alkaline phosphatase linked second antibody. of R2 mRNA levels in okadaic acid treated cells (Fig. 2A). This shows that an elevation in R2 protein alone resulted in increased enzyme activity (Table I), although both proteins are required for ribonucleotide reduction (Wright 1989), and supports previous findings that the R2 protein is the limiting component for ribonucleotide reductase activity in normal proliferating mammalian cells (Wright et al. 1990).
Effect of okadaic acid treatment on transcription of Rl and R2 genes The possibility that the increases in R1 and R2 message levels observed following exposure to okadaic acid were due to changes in gene transcription rates was tested by pretreating BALB c/3T3 cells with the transcription blocker actinomycin D (Phillips and Crowthers 1986), prior to exposure of the cells to okadaic acid. As shown in Fig. 5, actinomycin D (5 pg/mL) prevented the increase in R1 and R2 gene expressions previously observed following exposure to okadaic acid, suggesting that at least in part, okadaic acid increases ribonucleotide reductase gene expression by altering the transcriptional process. Effect of protein kinase C down-regulation on okadaic acid dependent RI and R2 mRNA induction Okadaic acid is a non-phorbol ester type tumor promoter (Suganuma et al. 1988) and it has no effect on any of the
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FIG. 5. Actinomycin D prevents the okadaic acid induced elevation of R2 and R1 message levels. (A) Northern blots of R2 mRNA levels in the absence of actinomycin D and okadaic acid (a); in the absence of actinornycin D, but with 10 nM okadaic acid for 1 (b), 4 (c), and 8 h (d); in the presence of actinornycin D (5 pg/mL) without okadaic acid (e); and in the presence of actinomycin D (5 pg/mL) with okadaic acid (10 nM) for 1 (A, 4 (g), and 8 h (h). (B) Northern blots of R1 mRNA levels as described above. (C) GAPDH loading controls of R2 and R1 blots. R2, R1, and GAPDH autoradiograms were exposed for 48, 96, and 24 h, respectively. protein kinases tested to date, including protein kinase C (Erdodi et al. 1988; Haystead et al. 1989). However, inhibition of protein phosphatases allows the unopposed activity of protein kinases constitutively present in the cell and leads to enhanced phosphorylation of many of the substrates of ~ r o t e i nkinases (Sassa et al. 1989). Previous observations from our laboratory (Choy et al. -1989) showed that there was a transient increase in R2 gene expression following treatment of BALB c/3T3 cells with the tumor promoter TPA. The mechanism of action of TPA involves modification of protein kinase C activity (Nishizuka 1986). Therefore, we were interested in evaluating the possibility that okadaic acid may be inducing alterations in ribonucleotide reductase gene expression through a mechanism that involves protein kinase C. To test this possibility, BALB c/3T3 cells were treated with 0.1 pM TPA for 48 h to downregulate protein kinase C activity (Young et al. 1987). Previous studies have indicated that R1 and R2 gene expressions are at approximately untreated control levels after 24-48 h of exposure to 0.1 pM TPA (Choy et al. 1989). Figure 6 clearly demonstrates that pretreatment of BALB c/3T3 cells with TPA for 48 h prevents the elevation of R1 and R2 message levels that are normally observed in the presence of okadaic acid. This result suggests a possible role for protein kinase C in the okadaic acid induced modulation of ribonucleotide reductase gene expression in BALB c/3T3 cells. Discussion Okadaic acid and calyculin A are potent inhibitors of protein phosphatase 1 and phosphatase 2A, two of the four major protein phosphatases in the cytosol of mammalian
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FIG. 6. TPA prevents the okadaic acid induced elevation of R2 and R1 message levels. BALB c/3T3 cells were treated with 0.1 pM TPA prior to exposure to 10 nM okadaic acid for various times. Northern blots are shown for R2 (A) and R1 (B) mRNA levels in the absence of okadaic acid but with 0.001% formamide (a); the presence of okadaic acid for 1 (b), 4 (c), and 8 h (d), respectively; in the presence of 0.1 pM TPA (48 h) and okadaic acid (1 h) (e); with TPA and okadaic acid (4 h) (A;and with TPA and okadaic acid (8 h) (g). R1 and R2 mRNA levels from cells treated with 0.1 pM TPA alone (48 h) are shown in (h). (C) GAPDH loading controls for the R2 and R1 Northern blots are also shown. Autoradiograms were exposed for 24-72 h at - 70°C with intensifying screens. The left-hand side is also shown in Fig. 2A; it is included here for ease of comparison.
cells that dephosphorylate serine and threonine residues (Cohen et al. 1990; Suganuma et al. 1990). It has been shown that okadaic acid does not inhibit protein tyrosine phosphatases or kinases that have been tested (Haystead et al. 1989). It appears that okadaic acid and calyculin A exert their effects by causing a net increase in the prevailing levels of phosphorylated proteins, an effect which is equivalent in many ways to activating protein kinases including protein kinase C. Results from this study indicate for the first time that treatment of mammalian cells with a protein phosphatase inhibitor can cause a significant increase in ribonucleotide reductase activity, R1 and R2 message, and R2 protein. It should be noted that ribonucleotide reductase is a highly regulated, rate-limiting step in DNA synthesis (Wright 1989; Wright et al. 1990), whose activity is normally coupled to the S phase of the cell cycle (Lewis et al. 1978; Thelander et al. 1985; Wright et al. 1990). The okadaic acid and calyculin A induced alterations in ribonucleotide reductase observed in this study occurred quickly (within 5 and 10 min, respectively), suggesting that a block at S phase or an unusual movement of cells into S phase is not responsible for the demonstrated modifications in ribonucleotide reductase activity and gene expression. These results are in keeping with DNA synthesis studies, which showed that there was no change in DNA synthesis rates in the presence of okadaic acid for even as long as 8 h, and with several recent investigations from our laboratory showing that mammalian ribonucleotide reductase gene expression can be elevated in the absence of detectable changes in DNA synthesis (Choy et al. 1989; Hurta
from the point of view of gaining a better understanding of DNA synthesis regulation, to determine more precisely the mechanism(s) responsible for this novel control of ribonucleotide reductase. Interestingly, the okadaic acid induced elevations in R1 and R2 message levels were blocked by treatment of cells with actinomycin D, indicating an involvement of the reductase transcriptional process. Previous studies in our laboratory (Choy et al. 1989) have demonstrated that ribonucleotide reductase activity and gene expression can be transiently modulated by the phorbol ester tumor promoter TPA, which activates protein kinase C (Nishizuka 1986). The pathways responsible for connecting the signal generated by protein kinase C activation by TPA and the alterations in ribonucleotide reductase gene expression are not known. Interestingly, down-regulation of protein kinase C with 0.1 pM TPA pretreatment abrogated the okadaic acid mediated elevation of ribonucleotide reductase mRNAs, consistent with the involvement of this signal pathway in the regulation of ribonucleotide reductase and the effects of okadaic acid. In agreement with this idea is the finding that TPA response elements are recognized, for example by the APl (jun/fos) transcription factor (Angel et al. 1987), and that protein phosphatases 1 and 2A appear to be involved as negative regulators of AP1 in human Jurkat T cells (Thevinin et al. 1991). Our observation that okadaic acid treatment of BALB c/3T3 fibroblasts lead to an elevation in message levels of a member of the jun family is also in keeping with this view. However, the specifics of the intracellular signalling pathway(s) responsible for okadaic acid and calyculin A modulation of ribonucleotide reductase gene expression and the role of protein phosphatases in this process remains to be further elucidated. Such investigations are in progress. In summary, this first demonstration that non-phorbol ester tumor promoters and protein phosphatase inhibitors can cause alterations in ribonucleotide reductase gene expression suggests that (i) ribonucleotide reductase, in particular the R2 component, participates in the critical early events involved in the process of tumor promotion, and (ii) illustrates a role for cellular protein phosphatases in the regulation of ribonucleotide reductase and, through this process, the regulation of DNA synthesis.
Acknowledgements Financial support to J.A.W. for this study was provided by the National Cancer Institute of Canada (NCIC), and the Natural Sciences and Engineering Research Council of Canada. J.A.W. is a Terry Fox Senior Research Scientist of the NCIC. R.A.R.H. is a previous recipient of a postdoctoral stipend from the Sellers Foundation, University of Manitoba. Angel, P., Imagawa, M., Chiu, R., Stein, B., Imbra, R.J., Rahmsdorf, H.J., Jonat, C., Herrlich, P., and Karin, M. 1987. Phorbol ester-inducible genes contain a common cis element recognized by a TPA-modulated trans-acting factor. Cell, 49: 729-739. Biolajan, C., and Takai, A. 1988. Inhibitory effect of a marinesponge toxin, okadaic acid, on protein phosphatases. Biochem. J. 256: 283-290. Choy, B.K., McClarty, G.A., Chan, A.K., Thelander, L., and Wright, J.A. 1988. Molecular mechanisms of drug resistance
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