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Experimental Therapeutics |
Department of Medical Oncology and Therapeutic Research, City of Hope National Medical Center, Duarte, California 91010 [L. X., B. Z., X. L., W. Q., Y. Y.], and Department of Pathophysiology, Zhejiang University School of Medicine, Hangzhou, Zhejiang 310031, People’s Republic of China [L. X., Z. J.]
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ABSTRACT |
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INTRODUCTION |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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), and hRRM2 and p53R2 are small protein subunits of RR (ß). The catalytically active form of eukaryotic RR is proposed to be a 2ß2 heterotetrameter made up of two large subunits (R1) and two small subunits (R2; Ref. 3
). Although the contribution of p53R2 to human cellular RR activity remains unclear (4)
, in vitro evidence in mice demonstrated that p53R2 forms an active ribonucleotide reductase tetramer with R1 (5)
. It is not clear whether human hRRM1 interacts with p53R2 in vivo in the same manner as it does with hRRM2. p53is a well-known tumor suppressor gene to which to two major biological processes have been attributed: cell cycle arrest and apoptosis (6, 7, 8) . p53 can function as a sequence-specific DNA binding protein and transcription factor for its target genes (9 , 10) . p53R2 has been identified as a transcriptional target of nuclear p53 (4) . It also has been shown that p53 can directly interact with other proteins in response to cellular stress (11 , 12) . It has been shown that hRRM2 protein is stabilized after UV irradiation, but transcription is not induced (13) . It is possible that p53 plays a role in this stabilization. Blocking of DNA synthesis by hydroxyurea, an RR inhibitor, was shown to stabilize p53, but it was extensively modified and its ability to induce transcription of many of its target genes was impaired (14) . A direct interaction between p53 and RR is hypothesized because RR itself is a target for cell cycle regulation. In this study, we hypothesize that p53 and RR directly interact to regulate RR activity required for response to DNA damage.
In the field of RR research, there are two opposing opinions about where deoxyribonucleotide synthesis occurs. The observations that rNDPs could be incorporated into DNA more efficiently than dNTPs and that enzymes associated with DNA metabolism were largely localized in the nucleus during S phase but were recovered in the cytoplasmic fraction and largely absent from the nucleus during G phase led Reddy and Pardee (15, 16, 17, 18) to suggest that RR and other DNA metabolic enzymes translocate from the cytoplasm to the nucleus and form a replitase to initiate DNA synthesis when cells pass from G1 to S phase of growth. However, subsequent immunocytochemical studies of both hRRM1 and hRRM2 showing localization in the cytoplasm of mammalian cells led other groups to argue against the replitase model (19 , 20) . Additional evidence has shown that the recently discovered hRRM2 homologue, p53R2, was found in that nucleus after DNA damage (21) but that hRRM1 remained in the cytoplasm of resting cells (20) . These findings led us to hypothesize that the RR subunits might traffic into the nucleus in response to DNA damage. However, nuclear accumulation of RR has never been directly demonstrated.
In this study, we have demonstrated that p53 directly interacts with p53R2 and hRRM2 but not hRRM1 and that hRRM1 interacts weakly with p53R2, hRRM2, or p53 in quiescent p53 wild-type KB cells. After exposure to UV, p53R2 and hRRM2 dissociated from p53 and bound hRRM1 in these cells. The PC3 cell line, which expresses a mutant form of p53 (22) , was also examined and did not show the shift in binding in response to UV. The interaction between p53 and the RR subunits was also altered in cells overexpressing hRRM2. We also clearly show the translocation of hRRM1, p53R2, and hRRM2 from the cytoplasm to the nucleus in response to UV irradiation, consistent with the increase in RR activity.
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MATERIALS AND METHODS |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Antibodies.
The rabbit polyclonal antibody against hRRM2 was a gift from Dr. Tim Kinsella’s lab. All other antibodies were obtained from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA). Mouse monoclonal and polyclonal antibody against p53, mouse polyclonal antibody against hRRM1, and goat polyclonal antibodies against hRRM2, p53R2, and hRRM1 were used for Western blotting. Agarose-conjugated normal goat IgG, agarose-conjugated p53 mouse monoclonal IgG2a, and agarose-conjugated hRRM1, hRRM2, and p53R2 goat polyclonal IgGs were used for IP. FITC-conjugated goat antirabbit, FITC-conjugated bovine antigoat, FITC-conjugated bovine antimouse, rhodamine-conjugated bovine antigoat, Texas Red-conjugated goat antimouse, and alkaline phosphatase-conjugated bovine antigoat IgG were used for the confocal studies.
IP and Western Blot.
Cells (107) were washed twice with PBS, lysed in 0.65 ml of ice-cold radioimmunoprecipitation assay buffer with freshly added inhibitors (1x PBS, 1% NP40, 0.5% sodium deoxycholate, 0.1% SDS, 100 µg/ml phenylmethylsulfonyl fluoride, 1 mM sodium orthovanadate, and 30 µl/ml aprotinin). The lysate was passed through a 27-gauge needle, debris was removed by centrifugation (10,000 rpm, 10 min., 4°C), and the total amount each of lysate was quantified (Bio-Rad protein assay). Ten mg of each lysate was precleared with agarose-conjugated normal goat IgG (30 min, 4°C) and subsequently incubated with agarose-conjugated hRRM1, hRRM2, or p53R2 goat polyclonal IgG or agarose-conjugated p53 mouse monoclonal IgG2a (15 µl, overnight at 4°C). Beads were collected by centrifugation (6000 rpm, 10 min, 4°C), and the immunoprecipitates were washed four times with lysis buffer (4°C) and solubilized in 60 µl of SDS-PAGE sample buffer. Seventy µg of each cell lysate and 12 µl of each immunoprecipitate were separated by SDS-PAGE and transferred to membrane. Fourteen percent SDS-PAGE was used for p53, p53R2, and hRRM2, and 10% SDS-PAGE was used for hRRM1. Immunoblotting was performed by incubation with the corresponding antibody (1:200 dilution, 1 h at room temperature) followed by incubation with alkaline phosphatase-conjugated goat antimouse (1:5000 dilution, 1 h at room temperature) or bovine antigoat (1:2000 dilution, 1 h at room temperature) secondary antibodies. Immunoreactive bands were visualized by enhanced chemiluminescence.
Confocal Immunofluorescence Analysis.
Cells were grown on sterile glass coverslips at 37°C for 24 h and UV irradiated as above. At the designated times, cells were washed briefly with PBS and fixed with 100% methanol for 5 min at -20°C. After air drying, fixed cells were washed with PBS three times, blocked for 2 h in a blocking buffer (10% BSA in PBS), and then additional incubated for another hour in PBS with 1.5% BSA containing anti-p53 mouse IgG, anti-p53R2 goat IgG, anti-hRRM2 rabbit IgG, or anti-hRRM1 mouse IgG. After washing three times in PBS, cells were incubated with Texas Red-, FITC-, or rhodamine-conjugated secondary antibodies in PBS with 1.5% BSA for 45 min. at 37°C in the dark. Coverslips were washed three times with PBS and mounted on microscopy cover glass with 90% glycerol in PBS. Images were acquired using a confocal microscope (Zeiss).
In Vivo RR Activity Assay.
Cells (1 x 106) were plated in a 60-mm dish and incubated for 24 h. Cells were irradiated by UV and harvested at the designated times. To enhance cell permeability, cells were washed twice in solution A [150 mM sucrose, 80 mM KCL, 35 mM HEPES (pH 7.4), 5 mM potassium phosphate (pH 7.4), 5 mM MgCl2, 0.5 mM CaCl2] and then suspended in 500 µl of cold solution A containing 0.25 mg/ml lysolecithin and incubated for 1 min at 4°C. To measure RR activity (ribonucleoside diphosphate reduction) and DNA synthesis, 1 x 106 permeabilized cells were incubated at 37°C for 10 min in a 300-µl reaction containing 50 mM HEPES (pH 7.4), 0.75 mM CaCl2, 10 mM phosphoenolpyruvate, 0.2 mM [3
H]rCDP, 0.2 mM rGDP, 0.2 mM rADP, and 0.2 mM dTDP. After incubation, reactions were divided into two equal volumes, mixed with 30 µl of 60% perchloric acid/0.1% Na PPi, and incubated on ice for 15 min. One ml of ddH2O was added, and samples were centrifuged to precipitate any acid-insoluble material. The pellet was extracted with 0.1 ml of 0.2 N NaOH and incubated at 37°C for 30 min. Seventy-five µl of each sample was suspended in 5 ml of Ecoscint A, and radioactivity was counted using a Beckman LS 5000CE liquid scintillation counter.
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RESULTS |
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shows that p53R2 and hRRM2 coimmunoprecipitate with p53 in KB cells but not in Hep3B cells, which do not express p53. The abundance of cellular p53R2 and hRRM2 was greater than that of p53 in KB cells (Fig. 1A
, Lane 4). This is consistent with previous reports that the level of p53 is low in unstimulated resting cells (23)
. Although hRRM2 and a low level of p53R2 were found in the Hep3B cell lysates (Fig. 1A
, Lane 2), they were not immunoprecipitated by p53 antibody (Fig. 1A
, Lane 1). Expression of hRRM1 was clearly detected in the cell lysates, but it was not detected in the immunoprecipitate. These findings indicate that p53 might interact directly with p53R2 and hRRM2 but not hRRM1. To additionally elucidate the functional significance of p53 binding to p53R2 and hRRM2 in vivo, DNA damage was induced in KB cells by exposure to UV. After UV irradiation, both p53R2 and hRRM2 dissociated from the complex with p53 (Fig. 1A
, Lane 5). The interaction between p53 and the small subunits of RR was confirmed by reciprocal IP experiments as shown in Fig. 1B
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, we performed co-IP assays using anti-hRRM1 antibody to examine the effect of UV exposure on RR complex formation (Fig. 1C)
. In the absence of UV irradiation (Fig. 1C
, Lane 1), a small amount of p53R2 was detected bound to hRRM1, whereas binding of hRRM2 was barely detectable. No co-IP of p53 was detected. After UV treatment, binding of p53R2 increased and an interaction with hRRM2 could now be detected (Fig. 1C
, Lane 3). The relative increase in protein binding to hRRM1 after UV treatment was much greater for hRRM2 than for p53R2.
Colocalization of p53 and p53R2 with hRRM2 in Wild-Type p53 KB Cells.
Confocal microscopy was used to show colocalization of p53 and the small RR subunits, providing additional evidence of interaction. KB cells were stained with anti-p53 and anti-p53R2 or anti-hRRM2 antibodies and are shown in Fig. 2
. p53 (green) and p53R2 and hRRM2 (red) appear distributed throughout the cytoplasm. The merged images show that p53 colocalizes with p53R2 and hRRM2 as indicated by the yellow color.
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. The localization of RR subunits was investigated in KB cells before UV treatment and at the indicated times after treatment with UV (12 J/m2). hRRM1, p53R2, and hRRM2 were detected in the cytoplasm of untreated KB cells (0 h). In UV-treated cells, accumulation of all three RR subunits in the nucleus could be detected after 1 h and increased after 2 h. The translocation of both hRRM2 and p53R2 from the cytoplasm to the nucleus in response to UV treatment suggests that both subunits may be activated to generate dNTPs required for DNA repair.
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. A significant increase in RR activity was detected 1 h after UV treatment, it peaked at 2 h, and it began to decrease after 3 h but still remained significantly elevated relative to untreated cells. The kinetics of RR activity shown in Fig. 3B
correspond closely to the time course of nuclear accumulation of RR subunits shown in Fig. 3A
. These results suggest that the increase in RR activity is related to the nuclear accumulation of RR subunits, which may be regulated by p53. It has been reported that although p53R2 and hRRM2 accumulate in the nucleus of p53-mutant PC3 cells, the increase in RR activity is delayed (24)
. This implies that the translocation of RR subunits from the cytoplasm into the nucleus and the associated increase in RR activity may be cell type-specific and depend on the intrinsic p53 status.
Mutant p53 Regulates RR in the Different Manner.
To additionally investigate how the status of p53 may influence the regulation of RR activity through shifting binding from p53 to hRRM1, PC3 cells, which express mutant p53, were investigated by co-IP as shown in Fig. 4
. In the absence of UV irradiation, both p53R2 and hRRM2 bind to mutant p53 (Fig. 4
, Lane 1) but with lower affinity than was seen for wild-type p53 in KB cells (as previously shown in Fig. 1A
, Lane 3). Binding of p53R2 and hRRM2 to hRRM1 (Fig. 4
, Lane 5) is similar in PC3 cells to that observed in KB cells (Fig. 1C
, Lane 1). After UV treatment, binding of p53R2 and hRRM2 to p53 was barely affected (Fig. 4
, Lane 3). Interestingly, the amount of p53R2 bound to hRRM1 did not increase in response to UV, whereas the amount of hRRM2 bound to hRRM1 increased slightly (Fig. 4
, Lane 7). These results indicate that mutation of p53 might cause a deficiency in the ability of hRRM2 and p53R2 to shift from binding p53 to hRRM1 in response to UV irradiation in p53 mutant PC3 cells.
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(25)
. The amount of hRRM2 protein in KBGem and KBHURs is increased 9.3- and 12-fold, respectively, compared with wild-type KB cells, whereas p53R2 and hRRM1 protein levels are unchanged (Fig. 5A)
. Activity of RR is increased 2.3- and 6.7-fold in KBGem and KBHURs, respectively (Table 1)
. The co-IP experiment shown in Fig. 5B
indicates that in KBGem and KBHURs cells the binding of p53R2 to p53 (Fig. 5
B, Lanes 1, 3, and 5) was decreased and binding to hRRM1 (Fig. 5B
, Lanes 7, 9, and 11) was increased relative to wild-type KB cells. This mimics the binding properties observed in wild-type KB cells after UV irradiation. However, in KBGem and KBHURs cells, the binding of hRRM2 to hRRM1 remains increased (Fig. 5
B, Lanes 7, 9, and 11), whereas the binding of hRRM2 to p53 is not significantly changed (Fig. 5
B, Lanes 1, 3, and 5). These results suggest that in cells overexpressing hRRM2, the affinity of p53R2 is decreased for p53 and enhanced for hRRM1. It is not surprising that the amount of hRRM2 bound to p53 was unchanged despite the increased binding to hRRM1 as the overall amount of hRRM2 in these cell lines is significantly increased. The aberrant binding of p53R2 to p53 and hRRM1 suggests that p53 may be involved in RR regulation.
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DISCUSSION |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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. Our results suggest that in quiescent cells, p53R2 and hRRM2 reside in the cytoplasm bound to p53. After cells are exposed to UV, p53R2 and hRRM2 dissociate from p53 and translocate into the nucleus where these small RR subunits bind to hRRM1 and form functional tetrameric RR holoenzyme, capable of synthesizing dNDPs. This process depends on wild-type p53.
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Regulation of RR activity through release of p53R2, allowing it to bind hRRM1 and form an active holoenzyme, is likely to induce nucleotide synthesis more rapidly than would a transcriptional regulation mechanism. The importance of p53 in DNA repair is supported by previous studies in which cells lacking functional p53 exhibited defective repair of UV damage and were more sensitive to UV irradiation than their wild-type p53 counterparts (4 , 35 , 36) . Inhibition of endogenous p53R2 expression in cells that have an intact p53-dependent DNA damage checkpoint reduced RR activity, DNA repair, and cell survival after exposure to various genotoxins (4) . In addition, it has been reported that transcription of p53R2 is induced by p53 in response to UV, and the increase in RR activity correlates with the p53R2 expression level after 48 h (4) . However, our results showed that there was no change of p53R2 expression in p53 wild-type cells 24 h after UV irradiation. This discrepancy in p53R2 regulation after UV irradiation might be because of differences in UV dosages, treatment times, or cell types. Direct supply of p53R2 to hRRM1 by release from a complex with p53 might facilitate rapid coupling between the p53 pathway and the RR activity needed for NER. This rapid mechanism might be followed later by a slower induction of transcription.
The other small subunit, hRRM2, has not been thought to play a role in DNA repair because studies in murine cells showed that levels of R2 decrease in G1 and G2 phases and only increase during S phase (37 , 38) . Others have suggested that R2 only supplies dNTPs for DNA replication in S phase, whereas p53R2 replaces it to supply dNTPs needed for DNA repair during G1 and G2 phases (4) . p53 contributes to the activation of G2 cell cycle arrest in response to DNA damage. However, cells transfected with p53R2 show only a slightly elevated number of cells in S-G2 phase after UV treatment (39) . This suggests that cell cycle arrest in response to DNA damage requires other signals in addition to p53R2 (4 , 37) . The role of R2 in repairing UV-induced DNA damage outside of S phase in yeast (40) , and the response of hRRM2 to ionizing radiation in human cervical carcinoma cells have been reported (41) . Although hRRM2 is known to be expressed during the cell cycle in S-G2 (42, 43, 44, 45) , our results demonstrate that p53 interacts with hRRM2 and releases it in response to UV, allowing it to bind hRRM1. Taken together, our results suggest that both p53R2 and hRRM2 are involved in DNA repair and that p53 regulates RR activity in response to DNA damage by direct interaction with the small RR subunits.
To address the question of where deoxyribonucleotides are synthesized in cells, we used confocal microscopy. Our results showed that p53R2, hRRM2, and hRRM1 translocated into the nucleus within 3 h of UV treatment, consistent with the timeframe for increase in RR activity. These results strongly suggest that synthesis of deoxyribonucleotides takes place in the nucleus. Nuclear synthesis of deoxyribonucleotides is also supported by the observation that permeabilized cells incorporated ribonucleoside diphosphates into DNA in preference to deoxyribonucleoside diphosphates. Moreover, in S-phase cells, dNTPs are concentrated in the nucleus (46)
. It has been shown that p53R2 localizes in the nucleus in a cell cycle-dependent and DNA damage-inducible manner (47)
. Our results are consistent with previous reports that hRRM1 and hRRM2 localize in the cytoplasm before stimulation (19
, 20) . Of interest, our study shows that p53R2 is also localized in the cytoplasm of quiescent cells. These findings suggest that trafficking of these three subunits into the nucleus provides RR enzyme activity for DNA repair. Arakawa et al. (21)
reported that p53R2, but not hRRM2, increased in nuclei 72 h after 
-irradiation. However, our results showed both p53R2 and hRRM2 translocated into the nucleus in response to UV. We have preliminary results (data not shown) demonstrating that hRRM2 returns to the cytoplasm within 3 h after cells are released from serum starvation. It is possible that after translocation in response to DNA damage hRRM2 returns to the cytoplasm rapidly, whereas p53R2 levels in the nucleus remain elevated for longer times. Alternatively the differences between our results and those of Arakawa et al. (21)
might be attributable to the induction of alternative DNA repair mechanisms after -irradiation.
UV irradiation of p53 wild-type cells induces modulation of the p53 level, cell cycle progression, and the occurrence of apoptosis. Disruption of p53 function was found to reduce repair of UV-induced DNA damage (36) . Here we have shown that UV-irradiated p53-mutant PC3 cells showed aberrant shifts in the binding of p53R2 and hRRM2 from p53 to hRRM1, indicating that mutant p53 could not regulate RR complex formation normally, which could lead to reduced DNA repair. Moreover, compared with KB cells, binding of p53R2 to hRRM1 was increased and binding to p53 was decreased in KBGem and KBHURs cells. It is possible that binding of p53R2 to p53 is decreased in these cells because all p53 binding sites are saturated by the high level of hRRM2 expression. The small RR subunits not sequestered by binding to p53 would be free to interact with hRRM1, leading to the observed increase in these cells. The mechanism of this finding requires additional elucidation.
We have shown here that UV irradiation induces the release of hRRM2 and p53R2 from wild-type p53 followed by binding to hRRM1 and translocation from the cytoplasm to the nucleus to form active RR holoenzyme. We have also shown that both hRRM2 and p53R2 might play a pivotal role in DNA repair. Understanding the molecular mechanism of RR regulation and the role of p53 in drug-resistant clones will have a direct impact on cancer therapy.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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1 Supported by NCI R01 Grant CA72767 
2 To whom requests for reprints should be addressed, at Department of Medical Oncology and Therapeutic Research, City of Hope National Medical Center, 1500 East Duarte Road, Duarte, CA 91010. Phone: (626) 359-8111, ext. 62867; Fax: (626) 301-8233; E-mail: yyen{at}coh.org
3 The abbreviations used are: RR, ribonucleotide reductase; NDP, nucleoside diphosphate; rNDP, ribonucleoside diphosphate; dNDP, deoxyribonucleoside diphosphate; dNTP, deoxynucleotide triphosphate; IP, immunoprecipitation; NER, nucleotide excision repair.
Received 6/21/02. Accepted 1/ 2/03.
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