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31 August 2000, Volume 19, Number 37, Pages 4283-4289
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Original Paper
A ribonucleotide reductase gene is a transcriptional target of p53 and p73
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Katsunori Nakano, Éva Bálint, Margaret Ashcroft and Karen H Vousden
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Regulation of Cell Growth Laboratory, NCI-FCRDC, Frederick, Maryland, MD 21702-1201, USA

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Correspondence to: K H Vousden, Regulation of Cell Growth Laboratory, NCI-FCRDC, Building 560, Room 22-96, West 7th Street, Frederick, MD 21702-1201, USA

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Abstract
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Many p53-inducible genes have been identified that might play a role in mediating the various downstream activities of p53. We have identified a close relative of ribonucleotide reductase, recently named p53R2, as a p53-inducible gene, and show that this gene is activated by several stress signals that activate a p53 response, including DNA damaging agents and p14ARF. p53R2 expression was induced by p53 mutants that are defective for the activation of apoptosis, but retain cell cycle arrest function, although no induction of p53R2 was seen in response to p21WAF1/CIP1-mediated cell cycle arrest. Several isoforms of the p53 family member p73 were also shown to induce p53R2 expression. Transient ectopic expression of either wild type p53R2 or p53R2 targeted to the nucleus, did not significantly alter cell cycle progression in unstressed cells. The identification of this gene as a p53 target supports a direct role for p53 in DNA repair, in addition to inhibition of growth of damaged cells. Oncogene (2000) 19, 4283-4289

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Keywords
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p53; p73; DNA damage; ribonucleotide reductase; p53R2

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Introduction

The p53 tumor suppressor gene plays an important role in preventing cancer development, and most tumor cells show either mutation in p53 or defects in the pathways responsible for the activation of p53 (Hollstein et al., 1999). p53 has been shown to participate in several cellular responses that could contribute to the suppression of tumor development, including DNA repair, senescence, cell cycle arrest and apoptosis (Ashcroft and Vousden, 1999). Activation of p53 in response to stress signals such as DNA damage or oncogene activation is thought to prevent the replication of abnormal cells and either allow their repair or target their elimination. In this way p53 can prevent the growth of cells that show uncontrolled proliferation or harbor potentially oncogenic mutations. The importance of p53 is emphasized by the dramatically enhanced cancer incidence in mice deleted of p53 (Attardi and Jacks, 1999), or humans with a germline mutation in one p53 allele (Evans and Lozano, 1997).

Understanding how p53 mediates its downstream effects has been of considerable interest. It is clear that one activity of p53 that plays an important role is the ability to function as a sequence specific DNA binding protein and transcription factor (Bates and Vousden, 1996; El-Deiry, 1998). Although several studies have suggested the existence of transcriptionally independent activities of p53, particularly in mediating the apoptotic response, in many systems activation of gene expression by p53 seems to be essential to mediate the p53 response (Bates and Vousden, 1999). Many p53 target genes have now been described, and in some cases it is possible to attribute p53-mediated responses to the activation of a known target gene. For example, activation of the cyclin-dependent kinase inhibitor p21WAF1/CIP1 (El-Deiry et al., 1993) leads to the G1 and G2 cell cycle arrest characteristic of p53 activation, but does not clearly participate in activation of the apoptotic response (Bates et al., 1998; Niculescu III et al., 1998). Deletion of p21WAF1/CIP1 substantially reduced p53-mediated cell cycle arrest (Waldman et al., 1995) (Bunz et al., 1998), but does not completely abolish this response, indicating that activation of other genes, such as 14-3-3 sigma (Hermeking et al., 1997), also contribute to the inhibition of cell cycle progression. Several excellent candidate genes for mediators of p53-dependent cell death have also been described, including Bax, Fas and DR5 (El-Deiry, 1998), although no single gene has been identified so far that could be considered the principal mediator of p53-induced apoptosis (Bates and Vousden, 1999). It therefore seems likely that each p53-induced response reflects the combined effects of activation of several target genes. We examined gene expression in response to p53 expression, and identified a novel p53 inducible gene encoding a protein closely related to ribonucleotide reductase. Ribonucleotide reductase is necessary for the production of deoxyribonucleotides and is important for both DNA replication and repair (Jordan and Reichard, 1998), and the identification of this new p53 target provides a direct mechanism through which p53 could contribute to DNA repair.

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Results and discussion

In order to identify novel p53 target genes that might mediate one or more p53 responses we generated a p53 null Saos-2 cell line inducible for expression of wild type p53, similar to the line described previously (Stott et al., 1998) (Figure 1a,b). Differences in gene expression in this line in the presence or absence of p53 were determined by analysing microarrays (Incyte) hybridized with cDNA derived from uninduced and induced cells. One of the tags identified was used as a probe in Northern blotting and detected an approximately 5.0 kb transcript whose expression was clearly enhanced following activation of wild type p53 in the inducible system (data not shown). 5' RACE was then carried out to isolate the full-length c-DNA. A novel 4.9 kb cDNA was obtained that contained a single open reading frame encoding a putative protein of 351 amino acids (Figure 2a). This protein showed a high degree of similarity to the small subunit of human ribonucleotide reductase, and very recently the same gene was independently isolated as p53 inducible by differential display, and termed p53R2 (Tanaka et al., 2000). To determine if activation of endogenous p53 would also regulate expression of this gene, we examined three wild type p53 expressing tumor cell lines (RKO, U2OS and MCF-7). Treatment of each of these lines with 5 nM actinomycin-D, which stabilizes and activates the endogenous p53 (Ashcroft et al., 2000), clearly induced expression of p53R2 (Figure 2b). The p53 dependence of this actinomycin-D response was confirmed by showing that expression of E6, which inactivates p53, resulted in loss of induction of this transcript in RKO cells (Figure 2b). Interestingly, the cell lines with endogenous p53 (RKO, MCF-7 and U2OS) all showed higher basal levels of p53R2 expression than the p53 null Saos-2 cells, and correspondingly higher levels of p53R2 expression following p53 induction was also seen in these cells (Figure 2b).

p53 has been shown to activate expression of a large number of genes and it has been suggested that different p53 targets might contribute to different activities of p53. Analysis of mutant p53 proteins that retain cell cycle arrest activity but fail to efficiently induce cell death suggests that p53 target genes that contribute to cell cycle arrest can be activated differentially from those that induce apoptosis (Friedlander et al., 1996; Ludwig et al., 1996). Analysis of several such p53 mutants indicated that while they retained wild type activity in the activation of transcription from the p21WAF1/CIP1 promoter, they showed a severe defect in the activation of other p53 responsive promoters such as Bax (Ryan and Vousden, 1998). To determine whether p53R2 was also differentially regulated in this way, we examined expression of p53R2 in cells inducibly expressing wild type p53, p53 175P, a mutant that retains cell cycle arrest activity but has severely diminished apoptotic function, and p53 175H, a mutant that fails to mediate either response (Figure 3). Each of the p53 proteins was expressed to similar levels following induction with doxycycline (Figure 3a). A Saos-2 line stably expressing p53 143A was also examined. The p53 143A mutant is temperature sensitive, showing loss of all activity at the non-permissive temperature (37°C), but acquiring the ability to activate cell cycle arrest, but not apoptosis, at the permissive temperature (30°C) (Friedlander et al., 1996). p53R2 expression was found to be activated in Saos-2 cells following induction of wild type p53 and p53 175P, and in cells expressing p53 143A that were incubated at the permissive temperature (Figure 3b,c). No activation of p53R2 expression was seen in response to p53 175H expression, or in the p53 143A cells grown at the non-permissive temperature.

These results raise the possibility that activation of p53R2 is related to cell cycle arrest, since wild type p53, p53 175P and p53 143A at the permissive temperature activate p21WAF1/CIP1 and block cells in the G1 and G2 phases of the cell cycle. We therefore examined the expression of p53R2 in Saos-2 cells inducibly expressing p21WAF1/CIP1, that show G1 and G2 cell cycle arrest indistinguishable from that induced by p53 (Bates et al., 1998). Activation of p21WAF1/CIP1 in these cells did not result in the elevation of p53R2 expression (Figure 3c), indicating that arrest of cell cycle progression per se does not lead to activation of p53R2.

Differential activation of p53 target gene expression can also be detected depending on the stress signal activating p53. Treatment of cells with actinomycin-D, for example, leads to elevated expression of two well established p53 targets, p21WAF1/CIP1 and MDM2. In contrast, treatment of the same cells with the topoisomerase inhibitor camptothecin (CPT), results in stabilization of p53 and activation of p21WAF1/CIP1, but no induction of MDM2 expression (Ashcroft et al., 2000). Given the potential importance of p53R2 in mediating the DNA repair function of p53, we examined the activation of p53R2 expression in response to various different stress signals (Figure 4). Although each treatment induced some increase in the expression of p53R2, actinomycin-D led to a higher elevation of p53R2 expression than CPT or UV treatment at this time point. This difference was not so clearly seen in the induction of p21WAF1/CIP1 expression, which was equivalent following actinomycin-D and UV treatment and only slightly lower in CPT treated cells (Figure 4a). Another way to stabilize p53 is through expression of p14ARF, a protein that binds MDM2 and prevents degradation (Sherr, 1998). Induction of p14ARF in a cell line expressing wild type p53 (Stott et al., 1998) leads to stabilization of p53 and transcriptional activation of both p21WAF1/CIP1 and p53R2 (Figure 4b,c). Taken together, these results show that diverse stress signals can activate p53R2 expression. Our observation that the level of activation of p53R2 expression depends on the signal inducing p53 might reflect a difference in the requirement for p53R2 in mediating the response to different forms of stress.

p53 has recently been shown to belong to a family of proteins including p63 and p73, although unlike p53 neither p63 nor p73 show clear activity as tumor suppressor proteins (Lohrum and Vousden, 1999). However, deletion of p63 or p73 in mice has shown that both proteins are important during normal development (Mills et al., 1999; Yang et al., 1999, 2000). To determine whether p53R2 might be induced by activation of p73 we analysed a series of Saos-2 cell lines inducible for expression of several isoforms of p73 (Figure 5a). Induction of p73alpha, beta and gamma expression led to the activation of p21WAF1/CIP1 expression (Figure 5a) as previously reported (Zhu et al., 1999). Northern blot analysis of these lines showed that p53R2 expression was also activated by each of the p73 isoforms (Figure 5b), although the levels of activation were somewhat lower than that seen following activation of p53. By comparison, p73alpha and beta activated p21WAF1/CIP1 mRNA as efficiently as p53 in this system, suggesting that the p73 proteins are slightly less efficient than p53 in the activation of p53R2. p73alpha has been shown to be activated by some forms of DNA damage through the activity of c-Abl (Agami et al., 1999; Gong et al., 1999; Yuan et al., 1999) and these results open the possibility that p53R2 might also function to mediate the cellular response to p73.

To examine the effects of p53R2 expression on cell growth, we cloned the full-length p53R2 cDNA with an N-terminal Flag tag to facilitate detection. Transient expression of this construct in p53 null Saos-2 cells, followed by flow cytometric analysis of transfected cells (selected by sorting for co-transfected CD20) showed only a very modest effect of p53R2 expression on the cell cycle profile (Figure 6a), with a small enhancement of cells in the S and G2 phases of the cell cycle compared to vector-only transfected cells. These results also indicated that p53R2 does not induce the apoptotic response, in agreement with a recent study (Tanaka et al., 2000). Our immunocytochemical analysis of the transfected cells showed that the p53R2 protein was expressed in both the cytoplasm and nucleus (Figure 6b). Tanaka et al. showed that p53R2 contributes to the activation of a G2 cell cycle arrest in response to DNA damage (Tanaka et al., 2000). This function of p53R2 was correlated with the translocation of p53R2 from the cytoplasm to the nucleus. We therefore generated a p53R2 protein containing a nuclear localization signal derived from SV40 LT, named p53R2nls. Although this protein was almost exclusively localized to the nucleus (Figure 6b), cells transfected with p53R2nls showed only the slight elevation of cells in S and G2 phases as observed following expression of wild type p53R2 (Figure 6a). The activation of a clear G2 arrest in response to DNA damage would therefore appear to require signals in addition to nuclear expression of p53R2.

We have identified a new p53 inducible gene encoding a ribonucleotide reductase. This gene was also identified recently by Tanaka et al. (Tanaka et al., 2000), and shown to play a role in the repair of DNA damage. We show that p53R2 is activated in response to stress signals, such as actinomycin-D treatment and p14ARF expression, that may activate p53 without causing DNA damage, indicating that p53R2 may have functions additional to those that contribute to DNA repair. The activation of p53R2 by p73alpha, beta and gamma also suggests a role for this protein in mediating the p53-independent DNA damage response pathway activated through c-Abl (Agami et al., 1999; Gong et al., 1999; Yuan et al., 1999). It will be of interest to determine whether activation of p53R2 is necessary for the developmental functions of p73 that were recently revealed by the generation of p73 deficient mice (Yang et al., 2000).

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Materials and methods

Plasmids

N-terminal hemagglutinin (HA)-tagged human p73alpha, p73beta or p73gamma full-length cDNAs were released from the plasmids pcDNA-HAp73alpha, beta and gamma, (obtained from G Melino, University of Rome, Rome, Italy) by digestion with HindIII, followed by blunt-end filling using Klenow, and digestion with XbaI. The fragments were ligated into the tet-responsive expression plasmid, pTRE (Clontech Laboratories, Inc.) at the EcoRI, (which was blunt-end filled using Klenow), and XbaI sites. Full-length human cDNA of p53 was subcloned into the pTRE plasmid digested at EcoRI and BamHI. The expression construct for 14ARF was described previously (Stott et al., 1998). The full-length Flag-tagged p53R2 was obtained by RT-PCR using sense primer, 5'TATGGATCCCGCACCATGGACTACAAGGACGACGATGACAAGGGCGACCCGGAAAGGCC'3 and anti-sense primer, 5'-TATGGATCCTTGGGAATATGCAGGGCGAG'3, then subcloned into pCMVneoBam to create pCMVneoBam-flag53R2. pCMVneoBam-flag53R2nls containing nuclear localization signal derived from SV40 LT at its C-terminus was also constructed by PCR using sense primer (as above) and anti-sense primer, TATGGATCCTCAGACCTTTCGCTTCTTCTTTGGAAAATCTGCATCCAAGGTGAAGACG and the pCMVneoBam-flag53R2 construct as a template. All PCR-derived constructs were confirmed by sequencing.

Cell lines, transfections and flow cytometry

MCF-7, RKO, RKO/E6 (stably expressing E6), U2OS, Saos-2, Saos2-143A (expressing the temperature-sensitive p53 mutant p53 143A), NARF2 (U2OS cells with inducible p14ARF) and Saos-2 cells inducible for p53 175P, p53 175H and p21WAF1/CIP1 were described previously (Stott et al., 1998; Bates et al., 1998; Ashcroft et al., 2000; Ryan et al., 2000). Saos-2 cells inducible for wild type p53 and various p73 isoforms were generated by transfection of the Saos-tet primary cell line (provided by K Ryan (Bates et al., 1998)) with 10 mug of pTREp53, pTREp73alpha, pTREp73beta, or pTREp73gamma, and 2 mug pSV2Hyg for stable selection of transfected cells. Cells were selected in hygromycin at 100 mug/ml for approximately 2 weeks. Independent clonal lines inducible for p53, p73alpha, p73beta, and p73gamma were analysed for inducible protein expression by Western blotting. All cell lines were maintained in DMEM supplemented with 10% FCS and grown at 37°C in an atmosphere of 10% CO2 in air. The temperature sensitive p53 was activated by moving cells to the permissive temperature (30°C). To induce protein expression the tetracycline inducible cell lines were treated with the tetracycline analog doxycycline at 2.0-2.5 mug/ml as indicated. p14ARF expression was induced in NARF2 cells by treatment with 1 mM IPTG. For induction of endogenous p53 protein in MCF-7, RKO, RKO/E6 (stably expressing E6), and U2OS cells, cells were treated with either actinomycin-D (5 nM), camptothecin (CPT, 2 muM), or UV (50 J/m-2) and harvested at the times indicated. Cells were transfected using calcium phosphate coprecipitation and harvested for either flow cytometry, or immuno-staining at the indicated times. For cell cycle analysis Saos-2 cells were transfected with 10 mug of total test plasmid and 1 mug pCMV CD20. Cells were harvested at 36 h after transfection, stained for CD20 expression and analysed by flow cytometry (FACScalibur, Becton Dickinson), as previously described (Rowan et al., 1996; Zhu et al., 1993).

Array screening and cloning of p53R2

Total RNA was collected from the p53 inducible Saos-2 cell line 24 h after treatment with or without 2.5 mug/ml doxycycline. The total RNA was sent to Incyte who isolated mRNA, generated cDNA and screened the human UniGEM V Microarray, and returned results showing fold changes in gene expression following p53 induction, using GEMTools software. One of the EST tags (GenBank accession number; AA768847) was identified and subjected for 5'RACE (SMART RACE, Clontech) according to the manufacturer's instructions to obtain the p53R2 cDNA.

RNA and protein analysis

Total RNA was isolated from cells grown to 70-80% confluency on a 15-cm diameter dish using TRIzol reagent (GIBCO-BRL), and 20 mug of total RNA per lane was examined by Northern blot analysis. 32P-dCTP-labeled full-length human p53R2 or p21WAF1/CIP1 cDNA probes were prepared using the Megaprime DNA labeling system according to the manufacturer's instructions (Amersham), and used for hybridization. The GAPDH cDNA probe (G3PDH) was purchased from Clontech. Western blot analysis was performed using total cell extracts as described previously (Ashcroft et al., 2000). Human p53, p21WAF1/CIP1 and beta-actin proteins were detected using the monoclonal antibodies DO-1, Ab-1 (Calbiochem), and C4 (Chemicon International Inc.), respectively. Human HA-tagged p73alpha, beta and gamma isoforms were detected using the F-7 anti-HA antibody (Santa Cruz Biotechnology, Inc.) or affinity purified rabbit polyclonal antibody that was raised against the C-terminal 19 amino acids of human p73beta (CTPPPPYHADPSLVRTWGP). The p14ARF polyclonal antibody has been described previously (Stott et al., 1998).

Immunostaining

Saos-2 cells were plated onto 10-cm diameter dishes (5´105 cells) containing 1-cm diameter sterile glass coverslips, and transfected as described above. Cells were washed three times with PBS, then fixed in 100% methanol for 10 min at room temperature. After fixation, cells were washed three times with PBS and blocked in PBS containing 0.5% BSA for 30 min at room temperature. Cells were incubated for 30 min at room temperature with M2 anti-flag antibody (at 1 : 500, Sigma) in blocking solution, washed three times with PBS, then incubated for 30 min at room temperature with a rabbit anti-mouse conjugated FITC antibody (at 1 : 100, DAKO) in blocking solution containing 1 mug/ml DAPI (Sigma). Cells were washed three times with PBS and slides were mounted with PBS/glycerol mount.

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Acknowledgements

We would like to thank Gerry Melino (University of Rome, Italy), for generously providing the human pcDNA-HAp73alpha, beta and gamma constructs, and Gordon Peters for the gift of NARF2 cells. We are extremely grateful to Andy Phillips, Kevin Ryan and all other members of the Vousden Lab for their invaluable help, advice and encouragement during the course of these studies.

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Figures
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Figure 1 Saos-2 cells with doxycycline inducible expression of wild type p53. (a) Flow cytometric analysis of Saos-2 cells (Saos-2p53) without treatment and after induction with doxycycline treatment (2.5 mug/ml) for 24 h, showing an increase of apoptotic cells with a sub-G1 DNA content, represented as a percentage (%) of the total population. (b) Western blot analysis of p53 expression in the inducible cells without treatment (-) and after induction with doxycycline (Dox) at 2.5 mug/ml for 24 h. p53 levels were assessed using monoclonal antibody DO-1

Figure 2 Expression of a newly identified p53-inducible gene in response to p53. (a) Amino acid sequence of the protein expressed by the p53-inducible gene. This gene has recently been named p53R2 by Tanaka et al. (Tanaka et al., 2000). (b) Northern blot showing expression of the identified p53-inducible gene (p53R2) in Saos-2 cells (Saos-2p53) without treatment (-) and after induction with doxycycline (Dox) at 2.5 mug/ml, and RKO cells, RKO/E6 cells, MCF-7 cells and U2OS cells treated without or with actinomycin-D (5 nM) for 24 h. GAPDH expression was examined as a loading control

Figure 3 Activation of p53R2 expression by p53 mutants. (a) Western blot analysis showing expression of p53 in Saos-2 cells following induction of wild type p53 (Saos-2p53), p53 175P, p53 175H without treatment (-) and after treatment with doxycycline (Dox) at 2.5 mug/ml for 24 h. Actin expression was examined as a loading control. (a) Northern blot analysis showing expression of p53R2 in Saos-2 cells following induction of wild type p53 (Saos-2p53), p53 175P, p53 175H without treatment (-) and after treatment with doxycycline (Dox) at 2.5 mug/ml for 24 h. GAPDH expression was examined as a loading control. (b) Northern blot analysis showing expression of p53R2 in Saos-2 cells following induction of wild type p53 (Saos-2p53), or p21WAF1/CIP1 without treatment (-) and after treatment with doxycycline (Dox) at 2.5 mug/ml, and in Saos-2 cells expressing the temperature sensitive p53 143A grown at the non-permissive (37°C) and permissive (30°C) temperatures. GAPDH expression was examined as a loading control

Figure 4 Activation of p53R2 expression in wild type p53 expressing cells exposed to p53-activating signals. (a) Northern blot analysis showing expression of p53R2 and p21WAF1/CIP1 in MCF-7 cells treated with CPT (2 muM), UV (50 J/m-2) or actinomycin-D (5 nM) for 12 h. GAPDH expression was examined as a loading control. (b) Expression of p53 and p14ARF in NARF2 cells (U2OS cells with inducible p14ARF) following activation of p14ARF by treatment with 1 mM IPTG for the indicated times. (c) Northern blot analysis of cells treated as in (b) showing p21WAF1/CIP1 and p53R2 expression in response to p14ARF. GAPDH expression was examined as a loading control

Figure 5 Activation of p53R2 expression by p73 isoforms. (a) Western blot analysis showing inducible expression of p73alpha, beta and gamma in Saos-2 cells without treatment (-) and after treatment with doxycycline (Dox) at 2 mug/ml. Induction of endogenousp21WAF1/CIP1 in response to p73alpha, beta and gamma is also shown. Actin expression was examined as a loading control. (b) Northern blot analysis showing expression of p53R2 and p21WAF1/CIP1 in response to p73alpha, beta and gamma induction without treatment (-) and after treatment with doxycycline (Dox) at 2.5 mug/ml. GAPDH expression was examined as a loading control

Figure 6 Expression of p53R2 in Saos-2 cells. (a) Flow cytometric analysis showing cell cycle profile of Saos-2 cells transfected with (10 mug) vector control, flag-p53R2 or flag-p53R2nls expression constructs. (b) Cellular localization of Saos-2 cells transfected with (10 mug) vector control, flag-p53R2 or flag-p53R2nls expression constructs. Localization of protein was detected using an anti-flag antibody and visualized using a FITC-conjugated secondary antibody. Cell nuclei were stained with DAPI

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Received 3 April 2000; revised 15 May 2000; accepted 4 July 2000
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31 August 2000, Volume 19, Number 37, Pages 4283-4289
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