The p53-regulated Cyclin-dependent Kinase Inhibitor, p21 (cip1, waf1, sdi1), Is Not Required for Global Genomic and Transcription-coupled Nucleotide Excision Repair of UV-induced DNA Photoproducts*

Shanthi Adimoolam, Cindy X. Lin, and James M. FordDagger

From the Departments of Medicine and Genetics, Division of Oncology, Stanford University School of Medicine, Stanford, California 94305

Received for publication, March 13, 2001, and in revised form, April 27, 2001


    ABSTRACT
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

The p53 tumor suppressor gene is a transcriptional activator involved in cell cycle regulation, apoptosis, and DNA repair. We have shown that p53 is required for efficient nucleotide excision repair of UV-induced DNA photoproducts from global genomic DNA but has no effect on transcription-coupled repair. In order to evaluate whether p53 influences repair indirectly through cell cycle arrest following DNA damage or plays a direct role, we examined repair in vivo in human cells genetically altered to disrupt or regulate the function of p53 and p21. Both primary human fibroblasts and HCT116 colon carcinoma cells wild type for p53 but in which the p21 gene was inactivated through targeted homologous recombination showed no decrease in global repair of UV photoproducts. Human bladder carcinoma cells mutant for p53 and containing a tetracycline-regulated p21 cDNA showed no significant enhancement of repair upon induction of p21 expression. All of the cell lines, including the mismatch repair-deficient, MLH1 mutant HCT116 cells, were proficient for transcription-coupled repair. Clonogenic survival of HCT116 cells following UV irradiation showed no dependence on p21. Therefore, our results indicate that p53-dependent nucleotide excision repair does not require the function of the p21 gene product and is independent of p53-regulated cell cycle checkpoints.


    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

The p53 tumor suppressor gene product is an integral and critical component of the mammalian cellular response to DNA damage, loss of which contributes to genomic instability and carcinogenesis (1). The molecular mechanisms involved in p53-dependent tumor suppression have been attributed to its well defined biological roles in cell cycle checkpoints and programmed cell death following DNA damage (1). Recently, we and others have shown that p53 is also involved in the repair of DNA damage, particularly in the nucleotide excision repair (NER)1 pathway (2-5). NER is a versatile and evolutionarily conserved DNA repair pathway with the ability to remove a wide range of DNA adducts induced by environmental and endogenous sources (6, 7). The most well studied of the lesions repaired by NER are the UV-induced cyclobutane pyrimidine dimers (CPDs) and [6-4] pyrimidine-pyrimidone photoproducts (6-4PPs). In humans, three inherited diseases are associated with defects in NER: xeroderma pigmentosum (XP), Cockayne's syndrome, and trichothiodystrophy (8). Patients with XP have a very high predisposition to skin cancer, whereas Cockayne's syndrome and trichothiodystrophy patients are not cancer-prone but exhibit developmental and neurological abnormalities. The mechanism of action of NER in eukaryotes has been well characterized and is attributed to a complex network of proteins that recognize the DNA adducts, produce dual incisions on either side of the lesion, facilitate removal of the excised oligonucleotide, and promote DNA resynthesis and subsequent ligation (9).

The process of NER can be divided into two genetically distinct pathways: the repair of lesions over the entire genome, referred to as global genomic repair (GGR), and the repair of transcription-blocking lesions present in transcribed DNA strands, which is known as transcription-coupled repair (TCR) (10, 11). The loss of p53 function specifically results in decreased GGR of CPDs but has no significant effect on TCR (2, 4, 12-15). This specific loss of GGR activity in p53-deficient cells suggests that the defect may involve proteins required specifically for GGR and not TCR, including those required for lesion recognition in genomic DNA, such as XPC; hHR23B, which binds to and stimulates XPC (16); and XPE (17). Although a role for p53 in NER has been well established, the precise molecular mechanism governing this effect is not completely understood. It has been suggested that p53 may regulate NER through protein-protein interactions (18), protein-nonspecific DNA binding (19), transcriptional control of downstream NER genes (14, 17, 20), or even indirect roles such as through cell cycle checkpoint functions (21).

The p53-regulated p21 gene (also known as waf1, cip1, or sdi1) is a cyclin-dependent kinase inhibitor and has been well documented as a mediator of the p53-dependent G1 cell cycle arrest following DNA damage (22). The G1-S phase cell cycle arrest occurs by the binding of p21 to cyclin-dependent kinases responsible for phosphorylation of cell cycle proteins such as the retinoblastoma protein that provide for entry into S phase (23). p21 also binds to proliferating cell nuclear antigen (PCNA) and inhibits DNA replication following damage by neutralizing the function of PCNA (24). It has been clearly demonstrated that p21 function is not required for p53-dependent apoptosis and that a different set of p53-transcriptionally regulated genes are responsible for the apoptotic activities mediated by p53 (25). However, the role of p21 in other p53-mediated biological processes, such as DNA repair, remains controversial. It has been suggested that p53 indirectly regulates DNA repair following DNA damage through a checkpoint function governed by p21, thereby "allowing time" for repair (21). If this hypothesis is correct, induced expression of p21 in the absence of wild type p53 or inhibition of p21 expression in the presence of wild type p53 should reproduce the effect of p53 function on NER. Due to different experimental approaches for assessing NER, significant controversy exists in the literature with regard to the potential role of p21 in NER in vivo. For example, using an exogenously introduced damaged reporter plasmid to assess in vivo cellular DNA repair, several groups have suggested that p21 is essential for NER (26, 27), whereas using in vitro repair assays others have found p21 dispensable for NER (28, 29). It has also been suggested that p21 may actually be inhibitory for NER (30, 31).

To ascertain whether the role of p53 in NER is through cell cycle arrest following DNA damage, we have evaluated the effect of p21 on NER activity in vivo systematically using a panel of both primary and transformed human tumor cell lines. To measure NER, we have utilized sensitive assays for UV-induced lesions in genomic DNA and in specific DNA sequences at various times following cellular irradiation. Here, we report results obtained with human cell lines wild type for p53 with heterozygous or homozygous knockouts of p21 and with human cell lines with mutant p53, allowing for inducible expression of p21. We present evidence that the absence of p21 has no effect on GGR or TCR and that overexpression of p21 in p53 mutant lines does not enhance NER. We conclude that the p53-regulated cell cycle protein, p21, is not required for either GGR or TCR. The role of p53 in regulating NER thus appears to be independent of its cell cycle checkpoint activity and is potentially mediated by directly regulating the expression and/or activity of target NER gene products.

    EXPERIMENTAL PROCEDURES
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Materials and Cell Lines-- Primary human LF1 fibroblasts, wild type, heterozygous, or homozygous for disruptions of the p21 gene, were a generous gift from Dr. John Sedivy (Brown University). The p21 knockouts were generated using a combination of novel strategies for efficient gene targeting in somatic cells and by establishing a culture system that enabled high single-cell cloning efficiency (32). LF1 fibroblasts were cultured in Ham's F-10 medium supplemented with 15% fetal bovine serum, G418, hygromycin B, and penicillin-streptomycin and grown at 37 °C in 5% CO2. HCT116 colon adenocarcinoma cells and its derivatives were obtained from Dr. Bert Vogelstein (Johns Hopkins University) (33) and grown in McCoy's 5A modified medium supplemented with 10% fetal bovine serum and antibiotics. HCT116 cells contain a homozygous truncating mutation in the human mismatch repair gene MLH1 (34). Derivatives of HCT116 carrying homozygous deletions of p21 or p53 were generated by a procedure similar to the one described by Brown et al. (32). EJ human bladder carcinoma lines and a derivative, EJp21, containing a tetracycline-regulated p21 transgene, were obtained from Dr. Stuart Aaronson (Mount Sinai School of Medicine). These cells contain both an oncogenically activated Ha-ras gene (35) and a mutant p53 gene with a mutation in exon 5 that renders it nonfunctional and incapable of transactivating the p21 gene. EJ cells were cultured in Dulbecco's modified Eagle's medium (high glucose) supplemented with penicillin-streptomycin and 10% fetal bovine serum. The EJp21 lines were additionally grown in G418 sulfate (750 µg/ml) and hygromycin B (100 µg/ml) to maintain integration of the two constructs containing the wild type p21 cDNA and the tetracycline-regulated transactivator element. Tetracycline (1 µg/ml) was added to the medium when suppression of p21 expression was required. The EJ and EJp21 lines were grown at 37 °C in 7% CO2. The list of cell lines and their genotypes are summarized in Table I.

                              
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Table I
Genetic properties of cell lines
The panel of cell lines used in this study is listed below along with the status of the p53 and/or the p21 protein function. MLH1 status is also indicated in the HCT116 lines.

Western Blotting-- The levels of p53, p21, MLH1, and MSH2 (mismatch repair) proteins in the various cell lines following UV damage were assayed by immunoblot analysis. Total cellular protein was obtained by lysing cells in a buffer containing 1% Triton X-100, 50 mM Tris·HCl, pH 8.0, 150 mM NaCl, 10 mM EDTA, 1 mM phenylmethylsulfonyl fluoride supplemented with the following protease inhibitors: 5 µg/ml leupeptin, 10 µg/ml pepstatin, and 100 µg/ml aprotinin. The lysed cells were centrifuged at 15,000 × g for 15 min, and the supernatants were collected. Protein concentrations were determined using the BCA protein quantitation kit (Pierce) according to the manufacturer's instructions. Equal amounts of total protein were separated by electrophoresis on 10% SDS-polyacrylamide gels (36) followed by electroblotting to nitrocellulose membranes (37). The membranes were blocked in TBS-Tween (TBS-T) with 2% Blotto and detected using mouse monoclonal antibodies to p53 (1:1000 in TBS-T-2% Blotto) (DO-1, Santa Cruz Biotechnology) and p21 (1:200 in TBS-T-2% Blotto) (SX118, PharMingen), followed by a 1-h incubation with a horseradish peroxidase-conjugated anti-mouse secondary antibody (1:1000 in TBS-T-2% Blotto). To confirm the MLH1 deficiency in HCT116 cells, anti-MLH1 (Ab1) and anti-MSH2 (Ab2) (Oncogene) antibodies were used. Anti-tubulin antibodies (B512, Sigma) were used to control for protein loading. Protein bands were detected using the Supersignal chemiluminescent substrate (Pierce) and autoradiography (Eastman Kodak Co.).

Global Genomic Repair Immunoslotblot Assay-- Repair of CPDs and 6-4PPs in the various cell lines at different times following UV irradiation was measured using an immunoslotblot assay (4). Briefly, exponentially growing cells were prelabeled with [3H]thymidine for ~48 h, washed with phosphate-buffered saline, and irradiated with 10 J/m2 UV using a germicidal lamp calibrated to deliver a dose of 1 J/m2/sec. The cells were lysed either immediately at 37 °C or after incubation in nonradioactive growth medium for various times following UV exposure. The lysis solution contained 10 mM Tris·HCl, pH 8.0, 1 mM EDTA, 0.5% SDS, 0.1 mg/ml proteinase K, and 0.1 mg/ml RNase. Genomic DNA was isolated by phenol/chloroform extraction, followed by ethanol precipitation, and the concentrations and specific radioactivity were determined. The genomic DNA was denatured and equal amounts from each sample in TE: 20× SSPE (1:1) were fixed onto Hybond N+ nylon membrane (Amersham Pharmacia Biotech) in triplicate using a slot-blot apparatus (100 ng of DNA for the detection of CPDs and 1 µg of DNA for the detection of 6-4 photoproducts). Genomic DNA from unirradiated cells was also loaded as a control. The membranes were blocked in 5% Blotto-TBS-Tween and then incubated with monoclonal antibodies specific for either CPDs (1:1000) or 6-4PPs (1:500) (obtained from Toshio Mori) (38). Horseradish peroxidase-conjugated secondary anti-mouse antibodies (1:3000 in phosphate-buffered saline), Supersignal chemiluminescence (Pierce) and phosphorimager detection (Bio-Rad) and analysis (Multi-analyst, Bio-Rad) were employed to detect and quantitate the UV-induced lesions. Data from triplicate DNA samples from three different biological experiments were averaged.

Transcription-coupled Repair Assay-- To determine the rate of removal of CPDs from the transcribed or nontranscribed strand of a specific gene fragment, strand-specific RNA probes were used to evaluate repair in a 20-kilobase pair KpnI restriction fragment spanning the central region of the DHFR gene (39). Cells were incubated with [3H]thymidine and UV-irradiated with 10 J/m2 (the dose required to introduce an average of one CPD per restriction fragment). The cells were lysed immediately after UV or incubated in nonradioactive growth medium containing 10-5 M 5-bromodeoxyuridine and 10-6 M fluorodeoxyuridine to density label the newly replicated DNA for various times and then lysed. Radioactive and density labeling permitted the isolation of unreplicated DNA by cesium chloride isopycnic density gradient sedimentation. Purified DNA was digested with KpnI. The KpnI-digested samples were treated or mock-treated with T4 bacteriophage endonuclease V (generously supplied by R. Stephen Lloyd, University of Texas-Galveston), which specifically recognizes CPDs. The T4 endonuclease V-digested DNA was electrophoresed in parallel under denaturing conditions, Southern transferred to a membrane, and hybridized with 30 × 106 cpm 32P-labeled strand-specific RNA probes generated by in vitro transcription of the plasmid pGEM0.69EH (39). The frequency of CPDs was calculated using the ratio of full-length restriction fragments in the T4 endonuclease V-treated and untreated samples and Poisson's distribution.

Clonogenic Survival Assays-- Exponentially growing cells were plated at very low cell densities (250-10,000 cells/100-mm dish) and exposed to UV irradiation at doses ranging from 0 to 20 J/m2. After culturing for 10-14 days, cells were rinsed in phosphate-buffered saline and fixed and stained by a 30-min incubation in 1% methylene blue in 50% ethanol/50% water. Colonies with greater than 50 cells were scored, and the plating efficiency and percentage of survival for each dose was calculated. Data from two different biological experiments were averaged.

    RESULTS
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Effects of p21 Knockout on NER Activity in Normal Diploid Fibroblast Cell Lines-- Initial studies on the effect of heterozygous and homozygous disruption of the p21 gene on GGR and TCR were performed in normal human fibroblasts. Knockouts of the p21 gene were generated by Brown et al. (32) by sequential gene targeting, first using a construct containing the neomycin-resistance gene, followed by a construct containing the hygromycin-resistance gene. The levels of p21 and p53 were assayed in the various cell lines following 15 J/m2 of UV irradiation. In the LF1 primary human fibroblasts wild type for p53, p21+/+ cells showed the expected UV-dependent induction of p21 levels, reaching a maximum at 48 h following UV (Fig. 1A). The p21 levels in the heterozygote p21+/- line were also induced by 16 h, peaking at 48 h. The p21-/- line had no immunodetectable levels of p21 protein, as expected. Up-regulation of the p53 protein was observed in all three cell lines (Fig. 1A).


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Fig. 1.   NER activity associated with loss of p21 in normal diploid fibroblast cell lines. A, primary LF1 fibroblasts wild type for p21 or containing homozygous or heterozygous deletions of the p21 gene were irradiated with UVC at a dose of 15 J/m2 and harvested at the various times indicated. Lysates were prepared and proteins detected by immunoblotting as described under "Experimental Procedures." p53 was detected using the DO-1 mouse monoclonal antibody (Santa Cruz Biotechnology), whereas p21 protein levels were detected using the SX118 mouse monoclonal antibody (PharMingen). B, exponentially growing cells were irradiated with 10 J/m2 UVC and incubated with growth media for 0-24 h post-UV to allow various times for repair. The cells were lysed at the indicated times and genomic DNA extracted. The relative amounts of CPD and 6-4 photoproducts compared with time 0 were determined using the immunoslotblot assay described under "Experimental Procedures." GGR of UV-induced CPDs (closed symbols) and 6-4PPs (open symbols) in LF1 primary fibroblasts wild type for p21 (black-triangle and triangle ), heterozygous ( and open circle ), or homozygous (black-square and ) knockouts of p21 are shown. The points shown are the average from three separate biological experiments, with each performed in triplicate. C, strand-specific RNA probes to the DHFR gene were used to assess the strand-specific removal of CPDs at various times following UV irradiation of cells with a dose of 10 J/m2. The lesion frequency at the respective times and the rate of removal of CPDs from the transcribed and nontranscribed strands of the DHFR gene were determined by quantifying the reappearance of specific full-length restriction fragments. Repair was assessed in the DHFR gene of LF1 primary human fibroblasts with heterozygous (black-square and ) or homozygous ( and open circle ) p21 knockouts. Repair in the transcribed strands are represented by the open symbols (open circle  and ), and the nontranscribed strands are represented by the closed symbols ( and black-square).

To evaluate the role of p21 in GGR, an immunoslotblot assay with monoclonal antibodies to CPDs and 6-4PPs was used to measure the removal of UV-induced lesions from total genomic DNA. In LF1, nearly 100% of 6-4PPs were removed by 4 h following UV irradiation, and ~40-50% of CPDs were removed by 24 h (Fig. 1B). This repair was similar in efficiency and kinetics to a number of other wild type primary human fibroblast cell lines previously examined (2, 4, 12). Because LF1 fibroblasts with somatic disruptions of the p21 gene were available, we were further able to assess the effects of loss of one or both wild type p21 alleles on GGR and compare it to repair in wild type primary parental fibroblasts. In fact, both p21+/- and p21-/- fibroblasts exhibited identical GGR of both CPDs and 6-4PPs compared with the p21 +/+ LF1 cells (Fig. 1B).

We have previously shown that p53 has no significant effect on TCR in a variety of cell lines (2, 4, 12, 14). Nevertheless, we wished to examine the potential effects of the p53-regulated p21 gene product on TCR to determine whether p21 played a role in TCR in a p53-independent manner. Using Southern hybridization and strand-specific RNA probes, we found that the repair of CPDs in the transcribed strand of LF1 p21+/- and p21-/- fibroblasts was similar to each other and to the repair observed in the transcribed strands of other primary fibroblast cell lines assayed previously (2, 4) (~75% by 24 h) (Fig. 1C). The nontranscribed strands in both the heterozygote and homozygote p21 knockouts were repaired to ~55-60%, which is the maximum repair observed for CPDs in repair proficient human cell lines.

The results from the LF1 fibroblast lines indicate that functional p21 is not required for normal levels of GGR or TCR after UV irradiation in human fibroblasts. Up-regulation of p53 following DNA-damage is intact in all three lines, suggesting a direct regulation of NER by p53, independent of p21.

NER Activity in Transformed Cell Lines with a Homozygous Deletion of the p21 or p53 Gene-- We next examined HCT116 human colon adenocarcinoma cell lines with somatic double knockouts of either the p53 or p21 gene, which in turn enabled us to assess the role of p21 in NER in a transformed phenotype. In the HCT116 p53+/+ line, immunoblot analysis demonstrated a UV-dependent up-regulation of the p53 protein starting at 2 h following UV irradiation, reaching peak levels at 8 h (Fig. 2A). Induction of the p21 protein was noted in the p53+/+ lines, whereas no such increase in p21 levels was observed in the p53-/- line. p21 expression was completely abolished in the HCT116 p21-/- line, although we detected normal UV irradiation-dependent induction of the p53 protein. Thus, the Western analyses confirmed that p21 expression is inducible in a UV and p53-dependent manner, when p21 is under the control of its p53-responsive endogenous promoter.


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Fig. 2.   Repair activity in HCT116 human colon carcinoma cells and its derivatives. A, HCT116 colon carcinoma lines wild type for p53 and p21, as well as homozygous knockouts of p53 or p21, were analyzed for induction of p53 or p21 protein levels post-UV. Antibodies used were as described in Fig. 1A. B, global genomic repair of CPDs (black-square, black-triangle, and ) and 6-4PPs (, triangle , and open circle ) in HCT116 cells wild type for p53 and p21 (black-square and ), p21 double knockouts (black-triangle and triangle ), or homozygous deletions of the p53 gene ( and open circle ) is indicated here. Points shown are the average from three separate biological experiments. C, strand-specific repair in the KpnI restriction fragment of the DHFR gene in HCT116 cells, wild type for p53 and p21 (black-square and ), p21-/- (black-triangle and triangle ), and p53-/- ( and open circle ), is shown for both the nontranscribed strand (black-square, black-triangle, and ) and the transcribed strand (, open circle , and triangle ).

GGR activity was measured in the HCT116 colon cancer cell lines, wild type for p53, and sublines containing either disruption of the p21 or the p53 gene. Similar to the LF1 fibroblast lines, these p53 wild type tumor cells exhibited efficient GGR of both 6-4PPs (~100% by 4 h) and CPDs (~60% repair by 24 h) (Fig. 2B). Furthermore, homozygous loss of p53 resulted in a decrease in GGR of CPDs (~30% repair by 24 h), as expected from our previous observations (2, 4). However, the HCT116 p21-/- cells showed no loss of GGR activity for either UV-induced CPDs or 6-4PPs, in accordance with results obtained with LF1 and its derivatives.

HCT116 wild type, p21-/- and p53-/- lines were also examined for TCR of CPDs. It has been previously reported that HCT116 colon cancer lines are mismatch repair-deficient due to a mutation in the MLH1 gene (34) and exhibit a defect in TCR (40, 41). Because we were interested in examining the role of p21 in TCR, we assayed for TCR in the HCT116 cells so as to determine whether subsequent knockout of p21 resulted in any additional defects on repair of either the transcribed or nontranscribed strands of the DHFR gene. However, contrary to previously published results (40, 41), we observed that the HCT116 cell lines were proficient for TCR (~90% repair in the transcribed strand and ~50% in the nontranscribed strand by 24 h) (Fig. 2C). Furthermore, HCT116 p21-/- cells exhibited TCR activity very similar to wild type, signifying that p21 played no role in modulating TCR. The strand-specific repair activity in the HCT116 p53-/- lines was similar to that observed previously in other p53-deficient lines (2, 4). Repair in the transcribed strand was similar to that in wild type (>80%), but repair in the nontranscribed strand was reduced (Fig. 2C). Western blot analysis using anti-MLH1 antibodies was used to confirm the MLH1 deficiency in the HCT116 cell lines (Fig. 3). HT1080 human fibrosarcoma cells are wild type for MLH1 and served as a positive control. Anti-MSH2 antibodies were used as controls because HCT116 cells are wild type for MSH2. As seen in Fig. 3, two separate wild type HCT116 cell lines derived from different clones were defective in MLH1 expression, thus verifying their mismatch repair deficiency.


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Fig. 3.   Expression of mismatch repair proteins MLH1 and MSH2 in HCT116 colon carcinoma cells. HCT116 cells were irradiated with 15 J/m2 of UVC, harvested either immediately or following a 24-h incubation, and total protein was extracted as described under "Experimental Procedures." HT1080 cells were used as a positive control for MLH1 expression. To detect MLH1 and MSH2, anti-MLH1 (Ab1) and anti-MSH2 (Ab2) mouse monoclonal antibodies were used (Oncogene). Tubulin (Sigma) was probed as a loading control.

Therefore, consistent with the results obtained with the LF1 lines, no role for p21 in NER was observed in transformed human cancer cell lines containing a somatic knockout of the p21 gene, whereas the role for p53 in GGR was confirmed in the p53 knockout.

Induced Overexpression of p21 in a p53 Mutant Background and Its Effects on NER-- In addition to the effects of complete disruption of the p21 gene, it was important to study the effects on NER of overexpression of the p21 gene in a p53 mutant background. For this purpose, we used the EJ human bladder carcinoma cell line homozygous for a nonfunctional p53 mutation, and its derivative EJp21, containing a tetracycline-regulatable human p21 transgene, allowing for the regulated overexpression of p21. In the EJ cells mutant for p53, low basal p21 levels were detected, and upon UV irradiation, an initial down-regulation was observed, but the levels returned to baseline by 24 h (Fig. 4A). No DNA damage-dependent induction of p21 above basal levels was noted. A similar expression profile was observed upon UV treatment in the EJp21 line containing a p21 gene under the control of the tetracycline-inducible promoter. In the presence of tetracycline, no differences were noted in p21 levels in the EJp21 line compared with the EJ cells. Significantly higher p21 levels were observed when tetracycline was removed at the time of UV, and p21 was dramatically overexpressed when tetracycline was removed 12 h prior to UV. Thus, in the absence of functional p53, or when p21 is under the control of the tetracycline-inducible promoter, an initial down-regulation was observed. This could be explained by transcription blocking lesions introduced into the p21 promoter or coding region, or due to UV-induced ubiquitination and/or degradation of p21 (42, 43).


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Fig. 4.   Effects of induced overexpression of p21 on NER in a p53 mutant background. A, EJ and EJp21 human bladder carcinoma cells were analyzed for p21 protein levels both when p21 expression was suppressed by the presence of tetracycline, as well as when p21 expression was induced either at the time of UV or 12 h prior to UV. The times indicated are incubation times following treatment with 15 J/m2 of UVC. B, global genomic repair of CPDs (black-square, cross , black-triangle, and ) and 6-4PPs (, , triangle , and open circle ) was measured in EJ bladder cancer cell lines mutant for p53, containing stable integration of the tetracycline-regulated p21 gene. GGR curves indicated are for EJ (mutant p53) (black-square and ), EJp21 (containing 1 µg/ml tetracycline (black-triangle and triangle )), EJp21 (with tetracycline withdrawal at the time of UV irradiation) ( and open circle ), and EJp21, with tetracycline withdrawal 12 h prior to UV (cross , ). The data represented here is the average from three separate biological experiments, with each performed in triplicate. C, preferential strand bias for repair in the DHFR gene is depicted in the EJp21 human bladder carcinoma cells mutant for p53 (with or without p21 expression). Repair of CPDs is shown in the nontranscribed (black-square, black-triangle, and ) and transcribed (, open circle , and triangle ) strands in the presence of tet (p21 expression suppressed) ( and open circle ), upon withdrawal of tet at the time of UV irradiation (black-square and ) and when tet was removed 12 h prior to UV (black-triangle and triangle ).

We next determined whether overexpression of wild type p21 in a p53 mutant cell line might enhance GGR. EJ bladder carcinoma cells mutant for p53 exhibit the predicted phenotype of poor CPD GGR (~15% at 24 h) (Fig. 4B). Similar to the parental EJ cells, EJp21 cells in the presence of tetracycline (see Fig. 4A for expression analysis) showed poor repair of CPDs. Induction of p21 following withdrawal of tetracycline at the time of UV irradiation (resulting in high p21 levels at 24 h) did not enhance GGR. However, when tetracycline was removed 12 h prior to UV, resulting in the presence of nonphysiologically high levels of p21 at the time of UV, a very slight improvement in GGR was noted. Repair of 6-4PPs was efficient in all cases with ~95% repair by 4 h.

Finally, strand-specific repair analysis was performed in EJp21 lines (Fig. 4C) to determine the TCR phenotype in these p53 mutant lines and whether overexpression of p21 alters repair of CPDs in the transcribed or nontranscribed strand of the DHFR gene. As shown in Fig. 4C, efficient repair in the transcribed strand was detected in the absence, as well as upon overexpression of p21 (~75%), even though functional p53 is absent. The repair in the nontranscribed strand of DHFR paralleled the results obtained with the GGR experiments, with essentially no repair observed in the absence of p21, or the presence of moderate p21 levels, but with slight improvement in repair when supraphysiological levels of p21 were present (20% versus 10% at 24 h).

The repair studies with the p53 mutant and p21-inducible lines demonstrate that 1) nonfunctional p53 disrupts normal GGR but has no effect on TCR, 2) expression of near physiological levels of p21 is incapable of rescuing the mutant p53-associated GGR defect, and 3) supraphysiological overexpression of p21 minimally enhances GGR but has no effect on TCR.

Influence of p21 Status on the Long Term Cellular Sensitivity of Cells to UV Irradiation-- Clonogenic survival assays were performed to determine the effect of p21 on the sensitivity of HCT116 cells to UV irradiation over an extended period of growth. Based upon the plating efficiencies of unirradiated cells (34% for p21+/+ and 8% for p21-/-), the percentage of survival was calculated and plotted versus the UV dose in J/m2 (Fig. 5). No difference in survival was observed in the p21-/- cells compared with p21 wild type HCT116 cells.


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Fig. 5.   Cellular sensitivity of HCT116 wild type and p21-/- cells to UV irradiation. Clonogenic survival following UV irradiation was determined for HCT116 wild type () and HCT116 p21-/- (black-square) cells. The curves shown represent the average of two separate biological experiments, each performed in triplicate.

Therefore, disruption of the function of the cdk inhibitor p21 in normal or tumor cells with wild type p53, or overexpression of p21 in a p53 mutant background, did not alter the GGR activity of the cells. Similarly, the presence or absence of p21 played no role on the cells ability to preferentially repair lesions in the transcribed strands of expressed genes compared with the respective nontranscribed strands. In addition to the absence of short-term effects on repair of UV-induced damage, no long-term effects on the survival to UV were observed in the absence of p21.

    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

The role of the p53-regulated p21 (also known as waf1, cip1, or sdi1) protein in cell cycle arrest, particularly in the G1 phase following DNA damage, has been well established, but its role in DNA repair remains controversial. Our results provide strong evidence that p21 is not required for the regulation of NER activity in vivo in human cells. We have measured global genomic repair of UV irradiation induced CPDs and 6-4PPs, as well as repair in the transcribed and nontranscribed strand of the endogenous DHFR gene. These activities were assessed in primary human fibroblasts and in transformed tumor cell lines with disrupted or regulated p21 expression. Our findings demonstrate that elimination of p21 as a p53 target does not disrupt p53-dependent inducible NER activity. Similarly, restoration of p21 expression in the absence of functional p53 does not significantly enhance or complement the NER defect associated with mutant p53. These results clearly signify that p21 is not a p53 target gene involved in NER.

Our primary focus has been to elucidate the mechanism of p53-dependent NER. We and others have previously demonstrated that functional p53 is required for efficient NER (2, 3, 13, 18). Specifically, p53 is required for global repair of UV-induced CPDs in the overall genome but is dispensable for the preferential transcription-coupled repair in the transcribed strand of expressed genes. The mechanism for p53-dependent NER has not, however, been elucidated. Several investigators have suggested that p53 may affect DNA repair through protein-protein or protein-DNA interactions. Wang et al. (18) demonstrated a direct interaction between the C-terminal domain of p53 and the transcription factor II-H-associated NER factors XP-B, XP-D, and Cockayne's syndrome B. Jayaraman and Prives (19) found that p53 is involved in binding to DNA single-strand ends and speculated that the excision product in NER activates p53 for DNA binding to p53 response elements. To study the mechanism of p53-dependent NER, we have analyzed the roles of several p53-regulated genes in NER. We have previously reported that p53 regulates the expression of the p48-XPE gene product (DDB2) and that XPE cells with mutant p48 exhibit the same NER-deficient phenotype observed in p53-/- cells (decreased GGR but normal TCR) (17). Furthermore, transfection and stable expression of the p48 gene in p53-/- cells allows for partial complementation of the repair deficiency (20, 44). Similar studies have indicated that gadd45, another p53-regulated gene, is also required for GGR, at least in murine cells (14). We have also recently found that p53 regulates the DNA damage induced expression of the human XPC gene product.2 Taken together, these observations strongly suggest that p53 regulates NER through transactivation of repair genes upon DNA damage.

In view of our interest in analyzing the role of p53-regulated genes in NER, and also to address the question of whether the role of p53 in NER is indirectly mediated via G1 cell cycle arrest following DNA damage, we examined the role of the p53-regulated cyclin-dependent kinase inhibitor p21 in NER. We used several cell lines with altered p21 expression that proved extremely valuable for our studies. Heterozygous and homozygous somatic disruptions of the p21 gene in LF1 primary human fibroblasts with wild type p53 provided a means of studying p21 effects on repair in a nontransformed phenotype, whereas homozygous p21 knockouts in HCT116 human colon carcinoma lines containing wild type p53 presented a transformed phenotype. These cell lines enabled us to evaluate whether p53-dependent NER was mediated by p21. A repair deficiency in the absence of p21 but with wild type p53 would provide a strong indication that p53 plays an indirect role in NER, which in turn may be mediated by its biological role in cell cycle arrest. We were also interested in determining any potential p21-regulated p53-independent effects on NER and therefore utilized the p53 mutant EJ bladder cancer cell line containing a tetracycline-regulated p21 gene. This cell line allowed us to regulate p21 expression in the absence of functional p53 and to determine whether p21 was capable of overriding the NER defect observed in this cell line. We confirmed using immunoblot analysis that the homozygous p21 knockout lines were completely devoid of immunodetectable p21, whereas the wild type p53 response was intact, as demonstrated by the DNA damage-dependent induction of p53. Similarly, strong repression of the tetracycline-regulated p21 promoter was achieved in the presence of tetracycline, allowing for the unambiguous analysis of the effects of p21 overexpression on NER.

We studied the in vivo NER activity using sensitive assays for GGR and TCR. To measure the repair of CPDs or 6-4PPs, we employed an immunoslotblot assay with monoclonal antibodies specific to each kind of UV-induced damage (4). We clearly demonstrate that in primary human fibroblasts or in tumor cell lines lacking functional p21, no difference in CPD or 6-4PP GGR activity is detectable compared with cells with wild type p21, whereas p53-/- HCT116 cells exhibit the same CPD repair defect reported previously by our laboratory (2, 4, 12, 15). EJ cells contain a mutation in exon 5 of the p53 gene that results in a nonfunctional p53 protein. GGR of CPDs in these cells is also significantly reduced, in fact to a greater extent than in HCT116 p53-/- cells (Fig. 2B). We studied the effects of tetracycline removal at the time of UV, which resulted in a more physiological representation of p21 levels, as well as removal 12 h prior to UV, which resulted in nonphysiologically high levels of p21 at the time of UV. In either case, no significant recovery of GGR was noted, further signifying that p21 plays no role in GGR activity following UV.

Despite the well established role for p53 in GGR, we have previously shown that disruptions in the p53 gene have no effect on transcription-coupled repair of the transcribed strand of expressed genes. Extrapolating these results, it is logical to assume that the presence or absence of functional p21 will have no effect on TCR. To address this theory, as well as to evaluate potential p53-independent effects of p21 on TCR, TCR activity was measured in several cell lines with variable p21 expression. Our results clearly demonstrate that p21 is not required for TCR. The rate of repair of CPDs in the transcribed strand in primary LF1 fibroblasts lacking functional p21 is identical to that observed in the normal fibroblast lines as shown previously (4). The rate of repair in the nontranscribed strand is similar to that observed using the GGR assay and lends further confirmation, as measurement of repair in the nontranscribed strand of the DHFR gene reflects the average repair capacity in the untranscribed regions of the genome. EJ bladder carcinoma cells mutant for p53 exhibit proficient repair of the transcribed strand, confirming our previous observations that p53 is not required for TCR, despite some recent contradictory results in the literature (45). Overexpression of p21 failed to significantly alter repair in either strand.

Our results demonstrating proficient TCR in the parental HCT116 cells are in contrast to those reported by Mellon et al. (40) and Leadon and Avrutskaya (41); both of these groups observed decreased TCR of UV-induced photoproducts in the same cells. This was thought to be due to mutation of MLH1, because correction of the mismatch repair defect through chromosome 3 transfer restored TCR activity in these experiments. Our present goal was to determine whether lack of functional p21 in the HCT116 cells resulted in further decrease in repair of the transcribed or the nontranscribed strand. We were surprised to note that HCT116 wild type cells exhibited normal TCR in our experiments (Fig. 2C) and thus confirmed by Western blotting that the HCT116 cells we used were MLH1-deficient. The reason for these contradictory TCR results with HCT116 wild type lines remains unclear. Although the HCT116 cell lines we used in our studies originates from the same line used in the other studies, it was subcloned during the process of creating the somatic gene knockouts (33). Given the intrinsic microsatellite instability associated with the mismatch repair deficiency in these cells, it is possible that the cell clones we have used have acquired a secondary mutation that either restores TCR or relieves an inhibition of TCR due to the primary MLH1 mutation. We are currently exploring this latter possibility with regard to the MutL heterodimeric partners of MLH1, PMS2 and PMS1, and the MutS genes MSH3 and MSH6, which are known to contain microsatellite sequences within their coding regions.

It can be speculated that the absence of p21 will result in elimination of the G1-S checkpoint, preventing cells from undergoing the normal cell cycle arrest and leading to unrestricted DNA replication. It has indeed been demonstrated in the LF1 fibroblasts that the p21-/- cells have lost their ability to arrest in G1 following gamma -irradiation (32). Similar loss of G1 blocks was observed with the HCT116 p21-/- cells when treated with DNA-damaging agents such as adriamycin or gamma  radiation (46). Cells lacking p21 appear to be capable of bypassing replicative senescence (32), which is in turn restored upon expression of p21 in p53 mutant cells (35). However, this clear defect in cell cycle regulation upon loss of p21 has no bearing on the NER capacity in these cells as evident from our current results. Nevertheless, the lack of p21 may result in varying rates of DNA replication following DNA damage. The assays that we employed for measuring GGR and TCR account for possible differences in DNA replication rates among various cell lines following UV irradiation. In GGR, prelabeling of cells with [3H]thymidine prior to UV enabled us to control for the amount of replication following UV and to ensure that equal amounts of unreplicated DNA were analyzed. This lends confidence that the observed results are not flawed by errors due to varying replication rates. TCR experiments eliminated any newly replicated DNA following UV irradiation by density labeling followed by cesium chloride gradients.

Our results, implying that p21 is not required for NER, are further supported by studies that examined the repair of UV-induced CPDs in the transcribed and the nontranscribed strand of the DHFR gene in human cells in different phases of the cell cycle: G1, early S, middle S, late S, and G2/M (47). The transcribed strand was preferentially repaired over the nontranscribed strand at all phases, and no significant cell cycle-dependent differences were noted in the repair activity. These studies signify that NER is equally efficient irrespective of the specific stage of the cell cycle in which a given cell population is, providing further credence to our observations that NER is not dependent on checkpoint function. Effects of other cell cycle checkpoint mediators, such as pRb on NER, have also been examined by assaying for repair in pRb-/- cells (14, 48). Similar to the results with p21, NER activity does not appear to be dependent on other cell cycle regulatory proteins. Our studies are also supported by the lack of tumor formation in mice with homozygous knockouts of p21 (49). Despite the defective G1 checkpoint control in these mice, other aspects of p53-regulated function, such as apoptosis and tumor prevention, appear normal. In fact, previously published results from our laboratory (14) also indicate that p21-/- murine embryonic fibroblasts are fully proficient in NER.

Several groups have previously analyzed the role of p21 in NER, primarily utilizing in vitro or host cell reactivation assays to measure repair, and varying results have been obtained. McDonald et al. (27) demonstrate that the same HCT116 cells lacking p21 that we have used had a 2-fold reduced capacity to restore reporter activity of UV or cisplatin exogenously damaged reporter plasmids. In addition, they showed enhancement of reporter host cell reactivation upon transient overexpression of p21 in p21-/- cells. Apparently, however, assaying for recovery of reporter gene expression does not reflect the excision repair capacity of these cells, because we found no decrease in in vivo GGR or TCR in these same cells. Thus, the differences observed may be attributed to potential differences in the ability of the host cells to promote transcription of a foreign reporter gene rather than differing repair capabilities. Also, host cell reactivation assays do not assess other DNA damage-inducible cellular responses, because the host cells are not irradiated. This is of major concern, because we now know that UV irradiation induces expression of several key NER gene products, such as p48-XPE, XPC, and Gadd45. Furthermore, although NER does appear to play a role in restoring UV-irradiated reporter gene expression, the level of reporter expression does not always correlate with removal of CPDs from the irradiated plasmid (50).

Clonogenic survival assays provide a means of assessing the cellular sensitivity to DNA damaging agents. The number of colonies obtained, relative to the number of cells plated, indicates the number of cells that survived a given dose of damaging agent and is termed the plating efficiency (PE) (51). The PE of the undamaged cells is set to 100%, and the percentage of survival under increasing doses of UV, for example, is obtained by the ratio of the PE at a particular dose to the PE of undamaged cells. We found that the plating efficiencies of unirradiated HCT116 p21+/+ and p21-/- cells were different, but the relative long-term survival responses after UV treatment are identical between the two cell lines. These results corroborate our repair data and strongly suggest that the lack of a difference in survival to UV is a consequence of the similarity in NER between the p21+/+ and p21-/- lines. McDonald et al. (27) report differences in clonogenic survival between the HCT116 p21+/+ and p21-/- cells that we have used. Although they state that p21-/- cells exhibited decreased clonogenic survival following UV irradiation than did p21+/+ cells, that study did not perform clonogenic survival experiments using standard methodology (51). What they actually measured was total cell number following a high lethal dose of UV (30-45 J/m2). Because they neither too into account differences in PEs nor quantitated colony formation, these results can only be interpreted to show a difference in cellular proliferation rates following DNA damage, likely due to the cell cycle effects of p21.

Using a similarly damaged reporter plasmid to assess NER, Sheikh et al. (26) and Wang et al. (42) also showed that inducible expression of p21 in a p53-defective background increased the repair capacity in these cells. We observed in our EJp21-inducible lines that supraphysiological overexpression of p21 prior to UV resulted in a slight enhancement of GGR. Because the above-mentioned studies did not segregate GGR and TCR activities, our results may serve to explain their observations, as most of those studies entailed overexpression of p21. Lack of p21 or physiological levels of p21 have no effect on NER, whereas overexpression of p21 might have some effect on NER. Further studies are required to resolve this observation. On the contrary, Pan et al. (31) and Cooper et al. (30) suggest that p21 overexpression actually inhibits NER by interacting with PCNA through the C-terminal domain of p21. However, these studies utilize in vitro repair assays using whole cell lysates to which p21 is added. Because the ratio of p21 to PCNA is significantly high, possibly active PCNA may be extracted from the reaction due to p21 binding, thereby inhibiting the repair reaction, as PCNA has been shown to be required for NER (52). However, Li et al. (29) and Shivji et al. (28) report no effect of p21 on NER with similar in vitro repair assays using whole cell lysates. Taken together, these studies demonstrate the difficulty in interpreting in vitro and host cell reactivation NER assays. In vivo genetic studies such as the studies we have described in our current paper are required to provide a more definitive answer.

In summary, we demonstrate that loss of p21 in a p53 wild type background or restoration of p21 in a p53 mutant background has no effect on either GGR or TCR. We conclude that the cdk inhibitor p21 is not a p53 target gene involved in NER. The mechanism of p53-dependent NER is independent of p21, and the presence or absence of cell cycle checkpoints does not correlate with NER activity. The role of p53 in NER is most likely due to the transactivation of a specific subset of downstream genes targeted to the NER pathway (p48-XPE, XPC, and gadd45 are likely candidates), and studies are currently under way in our laboratory to identify novel as well as known NER genes regulated by p53.

    ACKNOWLEDGEMENTS

We thank Drs. Phil Hanawalt and Ann Ganesan for helpful discussions and critical reading of the manuscript and Irina V. Cross for expert technical assistance. T4 endonuclease V was kindly provided by Dr. R. S. Lloyd.

    FOOTNOTES

* This work was supported in part by National Institutes of Health Award RO1 CA83889, by a Sidney Kimmel Foundation for Cancer Research Scholar Award, and by a Burroughs Wellcome Fund New Investigator Award in Toxicological Sciences.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Dagger To whom correspondence should be addressed: Dept. of Medicine, Division of Oncology, Stanford University School of Medicine, 1115 CCSR Bldg., Stanford, CA 94305. Tel.: 650-498-6689; Fax: 650-725-1420; E-mail: jmf@stanford.edu.

Published, JBC Papers in Press, April 30, 2001, DOI 10.1074/jbc.M102240200

2 S. Adimoolam and J. M. Ford, unpublished results.

    ABBREVIATIONS

The abbreviations used are: NER, nucleotide excision repair; CPD, cyclobutane pyrimidine dimer; 6-4PP, [6-4]-pyrimidine-pyrimidone photoproducts; XP, xeroderma pigmentosum; GGR, global genome repair; TCR, transcription-coupled repair; PE, plating efficiency; PCNA, proliferating cell nuclear antigen.

    REFERENCES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
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