Agents and Growth Arrest ... arrest. The same cell treatments that induced this p53 activity also caused an increase in cellular ..... turer (Tropix, Bedford, Mass.).
Vol. 13, No. 7
MoLEcuLAR AND CELLULAR BIOLOGY, JUlY 1993, p. 4242-4250 0270-7306/93/074242-09$02.00/0 Copyright © 1993, American Society for Microbiology
Induction of Cellular p53 Activity by DNA-Damaging Agents and Growth Arrest QIMIN ZHAN, FRANCE CARRIER, AND ALBERT J. FORNACE, JR.*
Laboratory of Molecular Pharmacology, DTP, DCT, National Cancer Institute, Room SCO9, Building 37, Bethesda, Maryland 20892 Received 4 February 1993/Returned for modification 15 March 1993/Accepted 9 April 1993
The tumor suppressor p53 can function as a sequence-specific transcription factor and is required for activation by ionizing radiation (IR) of one or more downstream effector genes, such as the human GADD45 gene. One important consequence of IR that is probably mediated by these downstream effector genes is activation of the p53-mediated G1 cell cycle checkpoint. While the induction of reporter constructs containing p53-binding sites has already been demonstrated with p53 expression vectors, we have now demonstrated the direct activation of such a construct after treatment of the human RKO line, which has a normal p53 phenotype, with various types of DNA-damaging agents and also after growth arrest produced by medium depletion (starvation). IR, UV radiation, and methylmethane sulfonate were found to induce p53 activity when a stably integrated reporter construct containing functional p53-binding sites was used and also in mobility shift assays with a p53-binding site from the GADD45 gene, and IR-inducible gene previously associated with growth arrest. The same cell treatments that induced this p53 activity also caused an increase in cellular p53 protein levels. The response in cells lacking normal p53 or in RKO cells expressing a dominant negative mutant p53 was markedly reduced. Interestingly, the spectrum of effective inducing agents for the above-described experiments was similar to that which induces GADD45 either in cells with a normal p53 status or, with the exception of IR, in cells lacking normal p53. These results indicate a role for p53 in the IR pathway, which is completely p53 dependent, and in other genotoxic stress responses, in which p53 has a cooperative effect but is not required. The tumor suppressor p53 gene encodes a nuclear phosphoprotein involved in the control of cell growth; it is the most commonly mutated gene found in human tumors (see reference 31 for a review). p53 can function as a sequencespecific DNA-binding protein that positively regulates gene expression. A 10-bp consensus sequence has been empirically derived, and this motif must occur as a repeat for effective p53 binding (8, 12). In addition, p53 can negatively regulate gene expression (29), probably by a direct interaction with other transcription factors, such as TATA-binding protein (28) and CBF (1). Increased expression of p53 or expression of wild-type (wt) p53 in tumor cells lacking this protein results in the inhibition of cell growth and in the accumulation of cells in the late G1 phase of the cell cycle (31). A role for p53 in the cellular response to genotoxic stress was suggested by the observation that the level of this protein increased in cells exposed to DNA-damaging agents (20, 24, 25). This protein was subsequently found to have a critical role in the activation of the G1 checkpoint after exposure to ionizing radiation (IR) (21, 24). The activation of cell growth delays or cell cycle checkpoints is an important response to DNA damage in both bacteria and eukaryotes; e.g., the sulA SOS gene encodes a growth arrest gene in Eschenichia coli and the RAD9 gene, a cell cycle checkpoint gene, in Saccharomyces cerevisiae (9). For human cell lines with a known p53 phenotype or primary cells from "knockout" mice containing either one p53 gene or no p53 genes, IR elicited an increase in p53 protein levels and activation of the G1 checkpoint only in cells with a wt p53 phenotype (20, 21, * Corresponding author. Electronic mail address: fornace@ ncifcrf.gov.
24). In cell lines containing a wt p53 gene(s) but expressing viral products, such as T antigen or mutant p53, both of which can block normal p53 function in a dominant negative manner (31), the normal response was also lost (21, 24). The loss of this p53-dependent checkpoint after IR exposure has important implications in carcinogenesis and mutagenesis because cells lacking a normal p53 phenotype progress into the S phase with unrepaired IR damage, while the G1 checkpoint in cells with a wt p53 phenotype allows time for repair prior to DNA replication (21). Recent evidence indicates that p53 is a critical component of a signaling pathway that is induced by IR and leads to activation of the G1 checkpoint (21). Cells from patients with the cancer-prone disease ataxia telangiectasia (AT) are deficient in the IR-activated G1 checkpoint and also in the induction of p53 protein after IR exposure. Another component of this pathway is the GADD45 gene, which is only inducible by IR in human and rodent cells with a wt p53 phenotype. AT cells have been found to be deficient in the IR induction of this gene (26). In support of a direct role for p53 in the induction of GADD45 was the finding of a conserved 20-bp sequence in the third intron of the human and hamster genes that corresponded to the p53 consensus sequence described above. This sequence from the human gene showed very strong in vitro binding to p53 protein. In mobility shift assays, nuclear extracts from gamma-irradiated ML-1 cells, which have a wt p53 phenotype, contained a factor that bound a 30-bp oligomer containing this sequence; appreciable binding was not seen in nonirradiated ML-1 cells or irradiated HL60 cells, which have a null p53 phenotype. This factor was found to contain p53, on the basis of the finding that the addition of p53 antibody resulted in a supershifted complex. The proposed pathway involves 4242
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EFFECT OF DNA-DAMAGING AGENTS ON p53 ACTIVITY
the following steps. (i) DNA damage is recognized by the AT gene product(s) or another factor that transmits the signal to the AT protein(s) at an early step in this pathway. (ii) p53 protein is induced by an as-yet-undefined translational or posttranslational mechanism requiring the AT protein(s). (iii) p53 acts as a transcription factor for downstream effector genes, including GADD45. (iv) One or more of these effector genes activates the G1 checkpoint. There is increasing evidence (reviewed in reference 17) that genotoxic stress elicits multiple responses in mammalian cells; the role of p53 in activating these responses remains to be determined. GADD45 was originally isolated from hamster cells and grouped with four other genes that demonstrated coordinate and rapid induction by agents producing high levels of base damage in DNA, such as UV radiation or the alkylating agent methylmethane sulfonate (MMS); the five GADD genes were also induced, but more slowly, by treatments producing growth arrest, such as medium depletion (starvation) (11). At relevant doses, IR produces relatively little base damage compared with MMS or UV radiation and only appreciably induced GADD45 in cells with a wt p53 phenotype (21); in contrast, the other agents were effective in all mammalian cells tested (9). These results indicate that the GADD45 gene may have two distinct responses to genotoxic stress: one specific for IR and p53 dependent and one specific for base-damaging agents and other growth arrest treatments (shared with the other GADD genes). However, induction by MMS was modestly reduced in AT cells as compared with normal cells (26), a result that may indicate a contribution of the AT-p53-IR pathway to this response. The induction of other DNA damage-inducible (DDI) genes, such as c-jun, the tumor necrosis factor (TNF) gene, and the collagenase gene, is similar to phorbol ester responses, and these genes are 12-0-tetradecanoylphorbol13-acetate (TPA) inducible, while GADD45 is not (17). Evidence has been presented that the induction of such genes is mediated by activation of the Src tyrosine kinase (6) or protein kinase C (17) via initial damage in the cell membrane. The purpose of the current study was to demonstrate direct transcriptional induction by DNA-damaging and other agents by use of a promoter-reporter construct containing p53-binding sites and to determine whether induction is specific for IR or whether induction also occurs for other agents that can activate the various responses described above. While such promoter-reporter constructs have been used to demonstrate induction by p53 produced by transfected p53 expression vectors (12, 22), induction produced by increased levels of normal cellular p53 has not been so demonstrated. The effect of various treatments in cells with known p53 phenotypes was examined by use of stably integrated and transiently transfected promoter-reporter constructs. In addition to IR, strong induction for the stably integrated constructs was also seen in cells with a wt p53 phenotype when a spectrum of agents that strongly induce the GADD genes was used. Induction was correlated with activation in nuclear extracts of binding to an oligomer containing the GADD45 p53-binding site and also with increased cellular p53 protein levels.
MATERIALS AND METHODS Plasmid clones. Constructs PG13-CAT, MG15-CAT, pC53SN3, p53-SCX3, and pCMVneo were provided by B. Vogelstein and M. B. Kastan. PG13-CAT contains 13 repeats of a p53-binding site inserted 5' to the polyomavirus early (basal) promoter linked to a chloramphenicol acetyltrans-
4243
ferase (CAT) reporter gene (22); MG15-CAT is a similar construct, but the repeat sequence has been altered so that p53 binding is lost (22). pC53-SN3 is a construct expressing the wt p53 protein driven by a cytomegalovirus promoter, while p53-SCX3 is a similar construct expressing the p53 protein with a substitution of Ala for Val-143 (2). For selection of stably expressed transfected DNA on the basis of G418 resistance, pSV2neo was used. pHG45-CAT2 was constructed by inserting the fragment from positions -909 to +144 of the human GADD45 promoter into the promoterless CAT construct pCAT-Basic (Promega); a detailed description of this construct will be published elsewhere (33). The CAT reporter construct -151Z-CAT was provided by C. Hatch and contained a portion (positions -151 to +98) of the human histone H2A.Z gene (16). The other CAT reporter constructs used and the promoter for each construct were as follows: pCAT-Control with a simian virus 40 promoter (Promega), pC15 CAT with a human immunodeficiency virus type 1 (HIV-1) promoter (13), p188 with a gibbon ape leukemia virus promoter (18), and actin-CAT with a chicken 1-actin promoter (27). Cells and cell treatment. The human colorectal carcinoma lines RKO and SW480 were obtained from M. B. Kastan (21), and the human large-cell carcinoma line H1299 was obtained from E. Sausville (21). Cells were grown in modified Ham's F12 medium with 10% fetal calf serum and treated with DNA-damaging agents as described previously (19, 26), except that IR was from a 137Cs source at 5.5 Gy/min (21). Unless otherwise specified, cells for all experiments were actively growing. MMS was from Aldrich, TPA (a gift from H. Nagasawa) was originally from Consolidated Midland Corp., tumor necrosis factor alpha (TNF) was from Sigma, interleukin 6 was from GIBCO-BRL, actinomycin D was from Sigma, and hydroxyurea was from Boehringer Mannheim. Plasmid DNA was transfected by the calcium phosphate method (5). In most experiments, stable integrants for the various constructs were used. For these experiments, cells were grown to 60% confluence in 100-mm dishes and cotransfected with S p,g of the CAT reporter construct and 0.25 p,g of pSV2neo; 36 h later, the cells were reseeded at a lower density in 175-cm2 flasks and incubated with 400 ,g of Geneticin (G418) (Sigma) per ml for 2 weeks with at least one medium change. Pooled cultures of at least 50 separate G418-resistant colonies were then collected and expanded for use. For one derived cell line (see Fig. 3), three different plasmids were introduced, at a ratio of 10 p,g of p53-SCX3 to 5 ,ug of PG13-CAT to 0.25 p,g of pSV2neo. For transient expression, RKO cells in 100-mm dishes were transfected with plasmid DNA, changed to fresh medium 16 h later, treated with DNA-damaging agents at 20 h after transfection, changed to fresh medium 4 h later for MMS-treated samples, and harvested for CAT analysis at 44 h after transfection. CAT assay. Cells were rinsed, suspended in buffered saline with a rubber policeman, centrifuged, and resuspended in 0.25 M Tris (pH 7.8). Cells were disrupted by three freezethaw cycles, and the supematant was collected following centrifugation. For inactivation of cellular acetylase activity, the solution was heated to 65°C for 10 min and then stored at -70°C. The protein concentration was measured with the Bio-Rad protein assay kit, and equivalent amounts of protein were used for each assay. CAT activity was determined by measuring the acetylation of 14C-labeled chloramphenicol by thin-layer chromatography as previously described (14). Radioactivity was measured directly on the thin-layer chromatography plates with a Betascope model 603 blot analyzer
4244
ZHAN ET AL.
(Betagen). Extracts from mock-transfected cells were used to obtain background activity. After this background activity was subtracted out, the specific CAT activity was calculated by determining the fraction of chloramphenicol that had been acetylated. The relative CAT activity was determined by normalizing the activity of the treated sample to that of the untreated sample. Each value represents the average of at least two separate determinations done on the same day. CAT activity was measured over the linear range of chloramphenicol acetylation such that the fraction acetylated was proportional to actual activity. Gel mobility shift assay. Nuclear extracts were prepared as described previously (4, 7). DNA-binding reactions were carried out for 20 min at room temperature in a buffer containing 20 mM N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid (HEPES) (pH 7.8), 100 mM KCl, 1 mM EDTA, 1 mM dithiothreitol, 1 Fg of poly(dI-dC) (Sigma), 104 dpm of labeled probe, 10% glycerol, and 15 Stg of nuclear protein extract in a volume of 30 ,ul. The probe used was a 30-mer double-stranded synthetic oligonucleotide containing the sequence TGGTACAGA T.TCTAAGCATjCTGG, which corresponds to positions +1569 to +1598 of the human GADD45 gene (21). In some experiments, unlabeled competitor DNA was included in the binding reactions and consisted of the same double-stranded DNA, except that the invariant C and G positions (indicated by underlining) were replaced with T and A, respectively. Each strand was labeled separately with T4 polynucleotide kinase (New England Biolabs) and [y-32P]ATP (5,000 Ci/mmol; New England Nuclear), and then the strands were annealed. Unincorporated counts were separated on a Nick column (Pharmacia) with 10 jig of poly(dI-dC) as the carrier. The samples were analyzed on a 4% nondenaturing acrylamide gel (4). p53 protein determination. For immunoblots, cells were washed in buffered saline and counted, and then cellular extracts were prepared as previously described (4), except that the volume was adjusted to 0.1 x 106 cells per pl for nuclear protein extraction. Nuclear extracts were mixed with an equal volume of 2x Laemmli sample buffer (21), resolved on a 7.5% sodium dodecyl sulfate-polyacrylamide gel, and transferred to nitrocellulose paper. Equivalent protein loading was verified by staining of the nitrocellulose membrane with the reversible dye Ponceau red. The membrane was destained, and Western blot (immunoblot) analysis was performed with a chemiluminescence detection system in accordance with the instructions of the manufacturer (Tropix, Bedford, Mass.). The first antibody was a mixture of the anti-p53 antibodies (Oncogene Science, Manhassett, N.Y.) Ab-1 (PAb421) and Ab-2 (PAb1801) at 0.1 ug/ml each. The second antibody was alkaline phosphataseconjugated goat anti-mouse immunoglobulin G and immunoglobulin M (Tropix); the substrate used was CSPD (Tropix). RESULTS AND DISCUSSION Effect of DNA-damaging agents and other treatments on a promoter-reporter construct containing p53-binding sites. Several human tumor cell lines were transfected with PG13CAT and assayed for DNA damage responsiveness. The RKO cell line has a wt p53 genotype and phenotype; the activation of the G1 checkpoint by IR and the induction of p53 protein by IR have been well characterized for these cells (21, 24). SW480 cells express only mutant p53 and contain one mutant allele and one inactive allele; these cells lack IR induction of p53 protein and GADD45 mRNA and IR
MOL. CELL. BIOL.
activation of the G1 checkpoint (21, 24). The H1299 cell line has a p53 null phenotype because of a homozygous deletion in the p53 gene (12). The CAT reporter construct PG13-CAT was used because its strong response to elevated p53 expression has been well characterized (22). For example, in cotransfection experiments with p53 expression vectors, CAT expression with this vector was approximately 50-fold higher with wt p53 than with mutant p53. Cells containing this construct, which was stably integrated in chromosomal DNA or transiently expressed, were UV irradiated and assayed for CAT activity (Fig. 1). As shown in Fig. 1 and Table 1, stably integrated PG13-CAT in cells with wt p53 showed strong induction. Clear induction was seen with the two lower doses, which produce minimal cytotoxicity, and induction was nearly maximal with the 15-J/m2 dose, which reduces clonagenic survival to approximately 10%. In contrast, the response was markedly lower in the two lines lacking wt p53; no appreciable induction occurred at the two lower UV doses, and the increase in the level of induction at the higher doses was approximately 1/10 that in RKO cells with wt p53. For SW480 cells, the slight effect might have been attributable to mutant p53, which these cells express at appreciable levels, but this explanation would seem to be unlikely for H1299 cells. These cells contain a small homozygous deletion in the proximal portion of the p53 gene and show no appreciable p53 expression. However, on long exposure, very faint staining with p53 antibody in the 53-kDa region of Western blots has been seen (Sa, 19a). The relative levels of CAT activity in untreated growing cells did not markedly vary among the three lines containing stably transfected PG13-CAT. It should be noted that the autoradiographic exposure times were varied for different rows in Fig. 1 to optimally demonstrate the relative effect of UV radiation and its dose dependence. The actual level of CAT activity in untreated RKO cells varied by twofold or less from that in the other two lines in all experiments (data not shown). It was also similar to that seen with MG15-CAT, indicating that the expression of this promoter in untreated cells was not dependent on p53. RKO cells containing stably integrated PG13-CAT were also responsive to treatments with MMS and IR, which produce different types of DNA damage repaired by different mechanisms (Table 1). At doses producing high levels of DNA base damage (17), MMS strongly induced this CAT construct nearly as well as UV radiation. As with UV radiation, the responses in the two cell lines lacking wt p53 were markedly lower. The response was much lower with IR than with UV radiation or MMS even at a dose, 20 Gy, at which there is essentially no clonagenic survival. Both the 5-Gy IR dose and the 15-J/m2 UV radiation dose reduce clonagenic survival in most cells to approximately equivalent levels, but the increase in CAT expression was more than 20-fold higher with the latter treatment. With IR in the other two cell lines, no increase in CAT activity occurred, in contrast to the small increase in activity with UV radiation or MMS. The significance of this difference is uncertain, considering that the increase with IR was only one- to twofold in RKO cells; if the response were reduced -10-fold in the H1299 and SW480 lines (as it was for UV radiation and MMS), then only a 0.2-fold increase (value of 1.2 in Table 1) might be expected compared with the actual values of 0.80 and 1.01 in Table 1. If the induction of PG13-CAT reflects cellular p53 activity, then relatively small increases will be required for activation of the G1 checkpoint, which will occur with the two lower IR doses used in Table 1 (20, 21, 24). Regarding GADD45 induction, the level of this mRNA
EFFECT OF DNA-DAMAGING AGENTS ON p53 ACTIVITY
VOL. 13, 1993
A Cell Line
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4245
in RKO cells increased 3.1-fold after a dose of 20 Gy (21), compared with a 2.9-fold increase in CAT expression in Table 1, while the level of this mRNA often increased more than 10-fold in a variety of mammalian cells after the same dose of MMS (10, 11). The responsiveness of PG13-CAT was assayed in both transient and stable transfection experiments because important differences between the two approaches have been seen with other DDI promoters in human cells. For example, we have confirmed the observations of Valerie and Rosenberg (30) that the HIV-1 promoter linked to a CAT reporter construct shows stronger UV induction in stable transfectants (unpublished data). It has been proposed that the enhanced DNA damage responsiveness may be due to changes in chromatin structure (30). Higher levels of induction also have been seen for GADD45-CAT and GADD153CAT constructs in stable transfectants (17). However, the difference with PG13-CAT was much more pronounced, with essentially no appreciable DNA damage responsiveness in transient transfection experiments. For example, the minimal effect in the transient transfection experiment with the highest UV dose was less than 1% that for the stable transfectants. The reason for this marked difference is uncertain but could involve several factors. One possibility is that normal cellular p53 is associated with chromatin such that the effective concentration is higher for the stably integrated construct. Another is that changes in chromatin structure after DNA damage, such as those proposed for the HIV-1 promoter-CAT construct after UV radiation (30), have an important role in the activation of certain DDI promoters. For ruling out nonspecific chromatin effects, a similar promoter-CAT construct containing mutated p53 sites, MG15-CAT, was treated in an identical manner, and no appreciable induction was seen in all experiments with the three cell lines (Fig. 1 and Table 1). The results with MG15-CAT also rule out the possibility of a DNA damageresponsive element in the basal polyomavirus promoter used in both constructs. Medium depletion has been found to be an effective growth arrest treatment that strongly induces the GADD genes and various GADD promoter-CAT constructs, although more slowly than DNA damage (10, 11). It has been our consistent finding that medium depletion (a type of starvation) is a much more effective inducing agent than other growth arrest treatments for the GADD genes in many mammalian cells. For example, in NIH 3T3 cells, the levels of the various GADD transcripts increased much more in medium-depleted confluent cells than in simply contactinhibited confluent cells; this result was in contrast to results seen with unrelated growth arrest genes (10). As with UV radiation and MMS, PG13-CAT was strongly induced in RKO stable transfectants by medium depletion, while the
.5 C-) 0
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UV Dose (J/m2)
FIG. 1. Effect of UV radiation on reporter gene constructs containing p53-binding sites. Cells containing stably integrated PG13-CAT or MG15-CAT were irradiated with the indicated dose; in the sample designated transient, RKO cells were transfected with 10 ,g of PG13-CAT 20 h prior to irradiation. Cells were harvested 24 h after irradiation, and CAT assays were performed for panel A as described in Materials and Methods. Autoradiographs in each row were from the same experiment and were obtained with the same autoradiographic exposure time. The dose response for the activation of CAT expression is shown in panel B. Values indicated by the broken line were derived from RKO cells stably expressing both PG13-CAT and p53-SCX3 (see Fig. 2).
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ZHAN ET AL.
MOL. CELL. BIOL.
TABLE 1. Activation of gene expression in human cell lines containing a CAT reporter construct and functional (PG13-CAT) or mutant (MG15-CAT) p53-binding motifs Fold increase in relative CAT activitya for the indicated cells with: PG13-CAT
Agent (dose)
RKO
Gamma ray (2 Gy) Gamma ray (5 Gy) Gamma ray (20 Gy) UV (2 J m-2) UV (5 J m-2) UV (15 J m-2) UV (30 J m-2)
God
MMS (50 tjg/ml)e MMS (100 p.g/ml)e TNF (100 U/ml) TNF (400 U/ml) TNF (1,000 U/ml) TPA (40 ng/ml)e Hydroxyurea (2 mM) Hydroxyurea (4 mM) Interleukin 6 (250 U/ml)
1.5 1.9 2.9 1.6 6.6 22.6 27.7 20.1 2.8 11.4 1 1.02 0.98 1.6 0.99 0.83 1.30
RKO
1.09 1.11 1.24
MG15-CAT
(mutant p53)c
H1299
SW480
RKO
1.12 1.17 3.2 9.0 10.8 8.8
0.80 1.14 1.34 3.0 3.8 1.6
1.01 0.84 1.20 2.0 2.2 1.11
0.87
4.8
2.9
2.1
1.30
H1299
SW480
1.16 1.21 0.99
1.04 1.18 1.31
1.22 1.09 1.16
1.1
1.35
1.39
a Results are taken from Fig. 1 and 2 and similar experiments. Relative CAT activity was normalized to that in untreated cells. Unless otherwise specified, cells were treated in the exponential growth phase, treatment with chemical agents was carried out for 24 h, and the reporter gene constructs were stably integrated in cellular DNA (see Results). Blank cells indicate that results were not determined. b RKO cells were transfected with PG13-CAT 24 h prior to treatment, as shown in Fig. 1. RKO cells also contained stably integrated p53-SCX3 as shown in Fig. 2. d Cells were grown to a high density such that they were confluent 2 days after the last medium change, and they were harvested 6 days after the last medium
change. e Treatment was carried out for 4 h, and cells were harvested 18 h later.
levels in H1299 and SW480 cells changed less than twofold (Table 1). As with the other treatments, there was no appreciable change in CAT activity with MG15-CAT in any experiment. If PG13-CAT induction reflects cellular p53 activity, then these findings indicate a role for p53 in another growth arrest state, which is also a type of stress (starvation). They also highlight a fundamental difference between how cells with wt p53 and cells with no p53 or with mutant p53 respond to a variety of adverse environmental conditions in the presence of which activation of growth arrest may be protective. The same cells were treated with a variety of agents that are known to activate various DDI and growth arrest genes but are ineffective for the GADD45 gene (Table 1). While many other DDI genes are phorbol ester responsive (9), TPA was an ineffective inducing agent for PG13-CAT in RKO cells, as was TNF, which is TPA or IR inducible and which can have a variety of cellular effects (15, 32). While DNA synthesis inhibitors, such as hydroxyurea, block DNA replication within minutes, they had no effect on mRNA levels of the five gadd genes for up to 8 h and only produced appreciable induction slowly. Treatment with DNA synthesis inhibitors for similar time periods also has been found not to cause appreciable increases in p53 protein levels (20). Although interleukin 6 can activate certain other growth arrest genes rapidly, this was not the case for GADD45, for which appreciable induction was not seen without several days of exposure (10). These negative results, taken together with other data in Table 1, show that the induction of PG13-CAT often correlates with treatments that rapidly induce GADD45 or, in the case of medium depletion, induce this gene over a longer time period. Suppression by expression of mutant p53 in RKO cells.
While the results obtained with the two cell lines lacking wt p53 indicate a role for p53 in the DNA damage responsiveness of PG13-CAT in RKO cells, more direct proof would be an alteration of the effective p53 level in RKO cells. It has been shown for RKO cells that the stable expression of a mutant p53 expression vector reduces the levels of p53 protein induction and GADD45 mRNA induction and suppresses activation of the G1 checkpoint after exposure to IR (21, 24). While the pCMV vector used shows strong expression in transient assays (22), we and another (19a) have found that stable expression often decreases with increasing number of passages of the cells, a result that perhaps is due to the progressive methylation of the stably integrated construct. Both polyclonal and monoclonal isolates of RKO cells containing this expression vector showed suppression of activation of the G1 checkpoint after exposure to IR; however, activation was not completely abolished, indicating that the levels of mutant p53 were not sufficient to totally block wt p53 action (24). For blocking PG13-CAT responsiveness, the same pCMV expression vector for mutant p53 as that used in the earlier studies was cotransfected with this CAT construct and pSV2neo; pooled cultures were used as soon as a sufficient number of cells were available. The level of induction of PG13-CAT in cells expressing mutant p53 was reduced with all three DNA-damaging agents and with medium depletion (Fig. 2 and Table 1). The level of induction was reduced at all UV doses, including the lower, nontoxic doses, and was between that in normal RKO cells and the two cell lines with 2 mutant p53 (Fig. 1B and Table 1). The relative reduction in CAT activity compared with that in normal RKO cells was similar for UV radiation, MMS, and medium depletion; the average relative induction for these treatments was 36.5% (standard deviation, +4.5%) that in
EFFECT OF DNA-DAMAGING AGENTS ON p53 ACIIVITY
VOL. 13, 1993
251
;0*pi
*PO
cO) *-0
'U 0-
U UV
MMS
G0
-y ray
Treatment FIG. 2. Effect of the expression of mutant p53 protein on the activation of PG13-CAT. RKO cells stably expressing PG13-CAT or PG3-CAT plus p53-SCX3 (see Materials and Methods) were treated and analyzed as described in the legend to Fig. 1. The results are shown diagrammatically, with cells containing PG13-CAT alone designated by solid bars and cells containing PG13-CAT plus p53-SCX3 designated by hatched bars. See Table 1, footnote d, for an explanation of Go.
matched cells not transfected with the mutant p53 expression vector. With IR, no appreciable induction occurred in cells containing mutant p53 (relative induction, 6%); however, the significance of this higher level of repression is uncertain, considering that IR was a weaker inducing agent than the other agents and that the actual values for IR were low. A reasonable interpretation of these results is that this dominant negative mutant p53 expressed in RKO cells markedly reduced PG13-CAT responsiveness by forming a complex with normal cellular p53 and preventing its binding to the p53-binding sites in this construct. The magnitude of the reduction in CAT activity was comparable to that of the abrogation of the G1 checkpoint; two-thirds of the RKO cells containing this mutant p53 construct failed to arrest and proceeded into the S phase after exposure to IR (24). The failure of this mutant p53 construct to completely block p53-type effects could have been due to a residual level of p53 multimer complexes containing only normal p53 protein. Recently, an E6 expression vector was shown to abrogate nearly totally this G1 checkpoint; expression of E6 has an advantage over expression of p53-SCX3 in that it causes the degradation of cellular p53, resulting in markedly reduced levels of this protein in RKO cells (23). Activation of the p53-binding site by DNA-damaging agents. To further support a role for normal cellular p53 in DNA damage responsiveness, we performed mobility shift assays with crude nuclear extracts from treated RKO cells. The oligomer used was the p53-binding site from the GADD45 gene; it bound baculovirus-produced p53 protein in vitro at least as strongly as the optimal sequence derived by screen-
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ing synthetic oligonucleotides and bound a cellular factor from gamma-irradiated wt p53 cells that also bound a p53 antibody (21). Figure 3A demonstrates that qualitatively similar binding activities were induced by all three DNAdamaging agents. The upper band was only clearly seen in extracts from treated cells, while the lower bands were also present in untreated cells and may have represented constitutive DNA-binding proteins. It should be noted that the induced band was qualitatively similar to that in gammairradiated ML-1 cells (data not shown). No appreciable binding activity was detected in any cytoplasmic extracts (data not shown) or in cells with a p53 null phenotype (21). The response was rapid, with strong binding activity in RKO cells being harvested in the first several hours after the start of treatment with UV radiation, MMS, or IR. With IR, the response in RKO cells was transient, with a clear decrease in activity by 3 h after irradiation, while the response was more prolonged with UV radiation and MMS. To further demonstrate that this binding was specific for p53, we conducted competition experiments with an unlabeled oligomer containing mutations at four invariant sites of the derived p53 consensus sequence (8, 12). This mutant sequence competed less effectively than unlabeled self sequence (Fig. 3B); similar results were also obtained in competition experiments with extracts from UV- or gamma-irradiated cells (data not shown). These results demonstrate that the same DNA-damaging agents that activate PG13-CAT also rapidly activate a nuclear factor that binds to a p53-binding site. The results obtained with a p53 antibody on the induced complex (21) and in the competition experiments (Fig. 3B) provide reasonable evidence that cellular p53 DNA-binding activity can be rapidly induced by DNA-damaging agents. Induction of p53 protein by different DNA-damaging agents and medium depletion. The evidence presented to this point strongly indicates that cellular p53 activity is induced by a variety of DNA-damaging agents but has not included the direct measurement of p53 protein. As discussed earlier, the activation of p53 by DNA-damaging agents is accompanied by an increase in the level of the protein. Therefore, p53 levels were determined by immunoblotting (Fig. 4) and were found to be increased in RKO cells by the same agents that induced PG13-CAT activity. The p53 protein doublet in this immunoblot was presumably due to a polymorphism that occurs in some individuals (20, 21, 24). The results shown here were obtained with nuclear extracts, but equivalent results were also obtained with whole-cell lysates. As with the mobility shift results shown in Fig. 3A, induction was rapid for UV radiation, MMS, and IR. The results shown in Fig. 4 indicate that p53 protein was rapidly induced by all three DNA-damaging agents; however, the response was more prolonged for UV radiation and MMS than for IR, as indicated by the mobility shift results shown in Fig. 3. The strong response to IR, as measured by mobility shift assays or p53 protein determinations, was in contrast to the weak response of PG13-CAT. There are several possible explanations for this difference. The simplest explanation would be that the difference is due to the fact that p53 is induced only transiently by IR, compared with a more prolonged induction by the other agents. Another possibility would be that some accessory factor is induced differently by IR than by the other agents. A third would be that there is some difference in p53 itself with the different agents; e.g., perhaps agents other than IR cause some posttranslational change(s) that produces increased activity. Regardless of which interpretation is correct, the finding of an increase in the p53 protein level provides direct evidence for a role of this
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ZHAN ET AL.
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B.
A. rn) 8 °
cf.)
10 50 100250
2i
>
o D)
SELF
E XX
MMS
MUTANT_ 10 +
50 100 250 -t
t
n Q 0
EL
LA'.
1111 I
-"
FREE
FREE
FIG. 3. Mobility shift assays with the GADD45 p53-binding site and extracts from RKO cells treated with DNA-damaging agents. Untreated cells (designated c) and cells treated with UV radiation (14 J m-2; 3 h), MMS (100 ,ug/ml; 4 h), or gamma rays (20 Gy; 1 or 3 h [designated Xl or X3, respectively]) were harvested at the indicated times after the start of treatment. (A) Nuclear extracts were incubated with labeled DNA and then analyzed by electrophoresis in a neutral polyacrylamide gel; nuclear extract was omitted in the sample designated Probe. (B) Competition experiments were performed with the indicated molar ratios of unlabeled DNA with the same sequence as the probe (SELF) or DNA containing mutations in the invariant G and C positions of this sequence (MUTANT) (see Materials and Methods). The arrows indicate the positions of the induced band; the positions of unbound freely migrating probe are designated FREE.
2p > U
p53--
-P:
X- ( i"""*dt :D
E
FIG. 4. p53 protein expression after treatment with DNA-damaging agents or medium depletion. RKO cells were treated as described in the legends to Fig. 1 and 3 and Table 1, footnote a, with the indicated agents and harvested 1 h after irradiation or the addition of MMS. Nuclear extracts from 2 x 106 cells were analyzed by immunoblotting with a p53 antibody (see Materials and Methods). Only the visualized bands are shown in the upper panel; their estimated size was 53 kDa. Equal total protein loading is illustrated by Ponceau red staining of the nitrocellulose membrane in the lower panel. See Table 1, footnote d, for an explanation of Go. C, control.
protein in the cellular response to a variety of stresses in addition to IR. As shown in Fig. 4 and 3, respectively, the magnitude of the increase in the p53 protein level was not as high as that seen in mobility shift assays with the same inducing agents. This result may reflect some qualitative change in p53 activity causing increased DNA binding in addition to the quantitative change. Another possibility would be the activation of some accessory factor that causes increased DNA binding. The increase in the p53 protein level after DNA damage has been shown to be due to some mechanism involving increased translation or increased protein stability, since p53 mRNA levels do not change (20, 21, 24, 25). Some qualitative change in this protein, induced by DNA damage or medium depletion, could very well increase both its stability and its activity. Role of p53 as a general suppressor of transcription. With activation of p53 by genotoxic stress, a second, more general effect on cells with a wt p53 phenotype would be the suppression of many cellular and viral genes by the interaction of p53 with common transcription factors, such as TBP (28) and CBF (1). Cotransfection of various cellular and viral promoters linked to the CAT reporter with a wt p53 expression vector markedly reduced their expression compared with that obtained with cotransfection with a control plasmid or a mutant p53 expression vector (Table 2). The ,-actin gene shows increased expression in growing cells compared with stationary cells (11) and was strongly inhibited by wt p53. Most of the cellular histone genes also show preferential expression in growing cells. For determination of whether the elements controlling basal expression were also affected by p53, a reporter gene containing only the proximal promoter of a mammalian histone gene was used (16); this
EFFECT OF DNA-DAMAGING AGENTS ON p53 ACTIVITY
VOL. 13, 1993
TABLE 2. Effect of wt and mutant p53 expression on the activation of CAT reporter genes containing various cellular and viral promoters Relative CAT Promoter
Plasmid
activity' pC53-SN3
GADD45
13-Actin
Histone Gibbon ape leukemia virus Simian virus 40 HIV-1 Mutant p53 p53
pHG45-CAT2 Actin-CAT
-151Z-CAT p188 pCAT-Control
pCl5 CAT MG15-CAT
with: p53-SCX3
0.05
0.93 1.16 1.15 0.79
0.06 0.06 0.35
0.98 0.93 1.26
0.30 0.13 0.10
62.4 0.98 PG13-CAT a RKO cells were transfected with 10 pg of the indicated CAT construct and 2 p.g of control plasmid (pCMVneo), wt p53 expression vector (pC53-SN3), or mutant p53 expression vector (p53-SCX3) and harvested to determine CAT activity 44 h later. Values indicate the CAT activity relative to that obtained with the control plasmid for each set.
promoter lacks the elements required for elevated expression in growing cells but was still found to be strongly inhibited by wt p53. Promoters from both DNA and RNA viruses were also strongly inhibited by wt p53. Results obtained with simian virus 40 and HIV-1 constructs confirmed previous observations (29) that these promoters are strongly inhibited by wt p53; however, we found no appreciable suppression of expression by the same mutant p53 that reduced expression twofold or more in the earlier study. In the case of the promoter for the human GADD45 gene, which is inducible by IR-activated p53 (21), transient expression of wt p53 also was found to inhibit a CAT construct containing this promoter; however, this promoter was not suppressed as strongly as the other tested promoters. The p53-binding element is contained in the third intron of this gene and presumably plays a role in its activation by p53 via an interaction with promoter elements. As seen previously with PG13-CAT (22), which contains p53-binding elements, cotransfection with the wt but not the mutant p53 expression vector increased CAT expression more than 50-fold, while no induction occurred with MG15-CAT. These results highlight another difference between how normal cells and tumor cells with an abnormal p53 status may respond after genotoxic stress and other growth arrest conditions, although it should be noted that p53 levels were probably much higher after transfection than those induced by biologically relevant doses of DNA-damaging agents. Speculation on some characteristics of the signaling mechanism(s) that involves p53 and that is activated by DNA damage. The spectrum of effective inducing agents for PG13CAT can be compared with that for different cellular responses to genotoxic stress. Excluding IR, agents inducing p53 activity show a striking similarity to agents inducing the GADD genes in mammalian cells, irrespective of p53 status. For example, in Chinese hamster ovary cells, MMS and UV radiation rapidly induced the five GADD genes, while DNA synthesis inhibitors and TPA did not (11); interleukin 6 also did not rapidly induce these genes in rodent cells (10). Over longer time periods, medium depletion has been found to be a strong inducing agent for the GADD genes in a variety of mammalian cells (9-11). In cells with a wt p53 phenotype, such as RKO, IR, even at very toxic doses, usually induced the GADD45 gene only two- to fourfold, as measured by
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mRNA levels (21), while larger increases in GADD45 mRNA levels frequently have been seen with MMS (9-11). Exceptions to this generalization are the findings in the myeloid cell line ML-1, in which the IR response was quite strong (21). In the current study, the level of induction of p53 activity, as measured with PG13-CAT, was higher with the base-damaging agents UV radiation and MMS than with IR. Activation was not dependent on the type of DNA repair, since damage caused by the base-damaging agents is repaired by different mechanisms (3). The similarity of PG13-CAT induction to that of the GADD genes is in contrast to other responses that are also induced by TPA (see earlier introductory comments) and by IR in many cells, irrespective of p53 status (17). There are several interpretations for the response of PG13-CAT in the SW480 and H1299 cell lines. While there was not appreciable induction by IR or medium depletion in these cells, a consistent small increase occurred after UV radiation and MMS; this increase was approximately 1 order of magnitude lower than that seen in RKO cells with wt p53. One possibility for this residual response may be the mutant p53 in SW480 cells and a very low level of p53 expression in H1299 cells (see earlier comments). A trivial explanation would be the presence of some DDI element in the downstream polyomavirus promoter, but this explanation is unlikely, considering that the MG15-CAT construct, which contains the same promoter, was unresponsive. Another possibility, which is difficult to exclude, is the presence of a second, unexpected "cryptic" responsive element in the p53-binding sites of PG13-CAT. An intriguing explanation would be the presence of a second cellular protein, perhaps related to p53, that is activated by base-damaging agents and binds to p53, with weak activation of this construct. Since the level of induction was much higher in cells with wt p53, a cooperative effect between p53 and this hypothetical second "factor" would be expected. p53 may well have a role in multiple genotoxic stress responses. With IR, induction of PG13-CAT and the GADD45 gene (21) has only been seen in cells with a wt p53 phenotype. Induction of p53 protein and the GADD45 gene also requires normal AT gene products (21). These observations indicate that the IR response involving GADD45 (and probably other downstream effector genes) is strictly dependent on p53 and the AT gene products (21). In contrast, strong GADD45 induction by MMS, UV radiation, or medium depletion occurs in cells lacking wt p53, and it has been proposed that this result indicates an alternate signaling pathway (21) (termed the MUM pathway now for brevity). However, induction by MMS was reduced somewhat in four AT lines compared with that in four normal lymphoblast lines for both GADD45 (P = 0.079) and GADD153 (P = 0.035) (26). In addition, both MMS and UV radiation strongly induced p53 activity in the current study. These results suggest that, while the response of GADD45 to the non-IR agents is not strictly dependent on p53, the AT-p53dependent pathway may contribute to this response. Several scenarios can be suggested to explain the similarities between these two pathways. (i) The similarity in the activation of p53 and the induction of the GADD genes, particularly GADD45, may be due to a common "trigger" of non-IR agents for both the AT-p53 and the MUM pathways; with IR, the initial triggering occurs at a later step that is specific for the AT-p53 pathway. (ii) In addition to p53, there is a "p53 accessory factor" that can activate the MUM pathway but can only weakly activate PG13-CAT (such as in SW480 and H1299 cells); presumably this factor would be induced by base-damaging agents but not by IR. (iii) Mutant p53 is
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activated by base-damaging agents sufficiently to induce the MUM pathway but not the AT-p53 pathway; however, this scenario does not explain GADD45 induction in cells with a p53 null phenotype, such as HL60 cells (11). (iv) The AT-p53 and MUM pathways are separate and independent, and the similarities between the two are due to the fact that they are partially redundant and that p53 can contribute to but is not required for activation of the latter pathway. Regardless of which scenario is involved, our results indicate a role for p53 in multiple genotoxic stress responses. ACKNOWLEDGMENTS We thank Michael B. Kastan for helpful discussions and for providing various cell lines and Bert Vogelstein for providing various plasmid constructs. REFERENCES 1. Agoff, S. N., J. Hou, D. I. H. LiAnzer, and B. Wu. 1993. Regulation of the human hsp70 promoter by p53. Science
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