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
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ABSTRACT |
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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.
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.
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.
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 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.
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
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
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
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
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
HCT116 wild type, p21
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).
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
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.
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 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 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 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 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 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 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.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
Genetic properties of cell lines
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.
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
/
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).
View larger version (21K):
[in a new window]
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 ( and
), heterozygous (
and
), or homozygous (
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 (
and
) or
homozygous (
and
) p21 knockouts. Repair in the transcribed
strands are represented by the open symbols (
and
),
and the nontranscribed strands are represented by the closed
symbols (
and
).
/
fibroblasts exhibited identical GGR of both CPDs and 6-4PPs
compared with the p21 +/+ LF1 cells (Fig. 1B).
/
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.
/
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.
View larger version (21K):
[in a new window]
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 ( ,
,
and
) and 6-4PPs (
,
, and
) in HCT116 cells wild type for
p53 and p21 (
and
), p21 double knockouts (
and
), or
homozygous deletions of the p53 gene (
and
) 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 (
and
), p21
/
(
and
), and p53
/
(
and
), is shown for both the
nontranscribed strand (
,
, and
) and the transcribed strand
(
,
, and
).
/
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.
/
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.
View larger version (44K):
[in a new window]
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.
View larger version (22K):
[in a new window]
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 ( ,
,
, and
) and 6-4PPs (
,
,
, and
) 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) (
and
), EJp21
(containing 1 µg/ml tetracycline (
and
)), EJp21 (with
tetracycline withdrawal at the time of UV irradiation) (
and
),
and EJp21, with tetracycline withdrawal 12 h prior to UV (
,
). 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 (
,
, and
) and transcribed
(
,
, and
) strands in the presence of tet (p21 expression
suppressed) (
and
), upon withdrawal of tet at the time of UV
irradiation (
and
) and when tet was removed 12 h prior to
UV (
and
).
/
), 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.
View larger version (14K):
[in a new window]
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
/
(
) cells.
The curves shown represent the average of two separate
biological experiments, each performed in triplicate.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
/
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.
/
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.
/
cells have lost their ability to arrest in
G1 following
-irradiation (32). Similar loss of
G1 blocks was observed with the HCT116 p21
/
cells when
treated with DNA-damaging agents such as adriamycin or
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.
/
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.
/
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).
/
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.
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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.
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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.
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.
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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.
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