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Molecular Biology and Genetics |
Department of Radiology [M. A. W., Q. Z. Z., M. E-M., S. V., A. A. W.], Biochemistry Program [S. V., A. A. W.], and James Cancer Hospital and Research Institute [A. A. W.], The Ohio State University, Columbus, Ohio 43210
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ABSTRACT |
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 Top ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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INTRODUCTION |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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It is well known that living cells rely on NER to remove a wide range of DNA damage caused by different physicochemical agents (19) . This defense system allows cells to cope with the harmful effects of various DNA-damaging agents (20) . An interesting feature of the excision repair process is its damage repertoire that allows different types of DNA lesions to be recognized and repaired (21 , 22) . The importance of DNA repair for maintaining normal cellular integrity and prevention of neoplastic phenotype is underscored by the fact that defective repair is linked to increased susceptibility of cells to toxic, mutagenic, and carcinogenic effects of genotoxin exposure (22 , 23) .
Benzo(a)pyrene is a major environmental pollutant and an ubiquitous carcinogen present in automobile exhaust, cigarette smoke, various foods, and industrial waste (24) . The diol-epoxide, (±)-anti-BPDE (racemic 7,8-dihydroxy-anti-9,10-epoxy-7,8,9,10-tetrahydro-benzo[a]pyrene), is the highly reactive electrophilic metabolite of benzo(a)pyrene and is reported to cause mutations, cytotoxicity, and inhibition of DNA synthesis in both prokaryotic and eukaryotic cells (25) . anti-BPDE reacts at several nucleophilic sites in DNA, and the covalent N2-dG-anti-BPDE adduct is most abundant (26) . It has been well established that the guanine adducts are the major adducts formed in cultured cells or DNA treated with racemic mixtures of BPDE, and 80–90% of the guanine adducts are the (+) optical isomers (27 , 28) . The DNA base alterations arising from exposure to (±)-anti-BPDE have been implicated in carcinogenesis and cellular transformation (29 , 30) . In the present report, we have used (±)-anti-BPDE as a model DNA-damaging agent to study the modulation of p53 induction response and its related effects in human cells having wild-type, mutant, or no p53 protein. We have shown that human fibroblast cells derived from normal individuals with wild-type p53 protein are more resistant to the cytotoxic action of anti-BPDE than cells with mutant p53 or no p53 protein derived from LFS patients. We have also shown the wild-type p53 dependence for efficient global genomic DNA repair as well as the removal of anti-BPDE-DNA adducts from the NTS. However, expression of wild-type p53 was not required for TCR of the transcribed strand, apoptosis, and the induction response of its signal transducer protein, p21.
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MATERIALS AND METHODS |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Cell Survival Assays.
Cell survival was measured using colony formation assay and MTT
metabolic viability assay. Exponentially growing cells were plated at
appropriate densities in 100-mm dishes and exposed to varying doses of
anti-BPDE after overnight attachment. The cells were then
allowed to grow for 14 days in growth medium, washed with PBS, fixed
with methanol:acetic acid (3:1), and stained with 0.1% crystal violet.
Colonies of >50 cells were counted after normalizing for plating
efficiency of cell types. For MTT assay, asynchronously growing cells
(5 x 103) were transferred into
96-well tissue culture plates (Corning) in 200 µl of complete medium.
After 24-h incubation, the medium was removed, the attached cells were
washed once with PBS, and the columns of 8 wells were exposed to
increasing doses of anti-BPDE. After 30-min carcinogen
treatment, cells were once again washed with PBS, replaced with fresh
medium, and incubated for another 24–72 h. Cell viability in
octuplicate wells was determined by the ability of live cells with
intact mitochondria to convert the soluble salt of MTT into an
insoluble formazan precipitate (35)
. Aliquots of 50-µl
MTT (1 mg/ml) were added to each well for 2 h, and the color
formed was quantitated by a spectrophotometric plate reader (Biotek)
after solubilization in DMSO. Cell viability was expressed relative to
the untreated control wells.
Western Blot Analysis.
Exponentially growing cells were washed with PBS and exposed to varying
doses of anti-BPDE. Cells were recovered by gentle
trypsinization after incubation for indicated time periods. The cells
were immediately lysed by boiling for 10 min in sample buffer [2%
SDS, 10% glycerol, 10 mM DTT in 62
mM Tris-HCl (pH 6.8), 10 µg/ml pepstatin, and
10 µg/ml leupeptin]. Western blot was performed with aliquots from
an equivalent number of cells, as described earlier (36)
.
Briefly, protein extracts from 
1.2 x 105 cells were separated in 8 or 12%
SDS-polyacrylamide gels and transferred to polyvinylidene difluoride
membranes using a semidry electroblotter (Hoeffer, San Francisco, CA).
Equal protein loading was confirmed from visualization of the membranes
stained for 15–20 min with fast green (0.1% fast green, 5% glacial
acetic acid, and 20% methanol). For p53 protein detection, a mixture
of anti-p53 protein antibodies [p53 Ab-2 and p53 Ab-6 from
hybridoma clones 1801 and DO-1, epitope maps within the
NH2 terminus (amino acid 32–79) and (amino acid
37–45) of p53, respectively] were used at 1:200 dilution. Antibodies
for the detection of p21 (clone DC60.2) were also used at a 1:200
dilution. All these antibodies were obtained from Neomarkers (Freemont,
CA). The membranes were developed after treatments with goat antimouse
IgG horseradish peroxidase conjugate (Boehringer Mannheim,
Indianapolis, IN). The specific proteins were visualized using the
enhanced chemiluminescence substrate reaction (Pierce, Rockford, IL).
All experiments were repeated at least thrice, and the results of
representative experiments are presented.
Quantitation of BPDE-DNA Adducts by Immunoslot Blot Assays.
The extent of anti-BPDE-DNA adduct formation and repair was
quantitated using noncompetitive immunoslot blot assay, as described
earlier (31)
. Briefly, cells were exposed to different
doses of anti-BPDE for 30 min. Cells were then washed with
PBS and either harvested immediately or maintained in serum-free medium
for varying posttreatment times. Cells were recovered by
trypsinization, and genomic DNA was isolated after immediate cell
lysis. DNA concentration was determined spectrophotometrically at 260
nm and by the microdiphenylamine assay (14)
. For
determining the extent of anti-BPDE-DNA adduct formation and
repair, increasing concentrations of unmodified, modified, and repaired
DNA samples were immobilized to nitrocellulose filters. The filters
were sequentially exposed to primary Pab BP1 and developed against
anti-BPDE-modified single-stranded DNA and enzyme-labeled
secondary antibody. The specificity of BP1 antibodies against various
modified DNA base adducts has been thoroughly characterized
(37)
, and their use in quantitative assessment of
anti-BPDA-treated cellular DNA has been described earlier
(38)
. The damage levels were calculated by comparing the
band intensities of the samples with anti-BPDE-modified DNA
standard samples run in parallel with all of the blots. The experiments
were repeated three times to ensure reproducibility of results.
DNA Fragmentation Analysis.
DNA fragmentation assays were performed essentially as described
earlier (17)
. Briefly, at various posttreatment times, the
attached cells were gently trypsinized, mixed with any unattached
cells, removed with the medium, and pelleted by centrifugation. DNA was
isolated from the cell pellets by a differential cellular lysis
procedure that separates fragmented DNA released only by apoptotic
cells from native intact DNA of unaffected cells by centrifugation. The
13,000-g supernatant was treated with RNase-A for 30 min at 37°C
followed by Proteinase K digestion for 1 h at 45°C. Supernatant
sample aliquots, equivalent to 1 x 106 cells, were separated by agarose gel
electrophoresis (1.2%) and visualized by ethidium bromide staining.
The apoptotic index was calculated based on the ratio of the fraction
of released fragmented DNA:the total DNA in the initial pellet
(13)
.
Strand-specific Repair Analysis by UvrABC Excision Reaction.
Strand-specific repair of anti-BPDE-DNA adducts was examined
within the TS and NTS of the 16-Kb EcoRI restriction
fragment containing all of the exons of the p53 gene except exon-1. The
UvrA protein was purified from Escherichia coli SURE
host strain harboring a UvrA expression vector, pUNC45, as described
(39)
. UvrB and UvrC proteins were purified from E.
coli XL-1 Blue and C41(DE3) strains containing vector pUTG97 and
pUTG98 respectively, using the intein-mediated purification with
an affinity chitin-binding tag system. Approximately 40 µg of DNA,
from cells, treated with 2 µM
anti-BPDE and allowed different periods of time to remove
BPDE-DNA adducts, was digested with EcoRI (5 units/µg of
DNA) at 37°C for 4–6 h. Complete digestion was verified by
electrophoresis of sample aliquots on agarose minigels. The digested
DNA was purified by extraction with Phenol/Chloroform (3.5:6),
precipitated with ethanol, and quantitated. Five pg of linearized
plasmid DNA containing the p53 cDNA sequence was added into each DNA
sample as an internal standard. One-half of the mixture was exposed to
UvrABC excinuclease, and the other half was exposed to reaction buffer
alone under conditions of excinuclease digestion. The reaction
mixture contained 20 µg of EcoRI-digested genomic DNA plus
10 pmol of UvrA, 50 pmol of UvrB, and 50 pmol of UvrC in a final volume
of buffer containing 50 mM Tris-HCl (pH 7.5), 10
mM MgCl 2, 75
mM KCl, 2 mM ATP, 1
mM DTT, and 1 µg/µl BSA. The samples were
incubated at 37°C for 1 h, and the reaction was stopped by
adding 0.1 µg/µl Proteinase K and 0.1% SDS to each sample followed
by an additional incubation at 42°C for 1 h. The UvrABC-treated
DNA was purified by extraction with phenol/chloroform (3.5:6) and
precipitated with ethanol. The samples were then dissolved in
denaturation buffer [90% formamide, 0.025% bromphenol blue] and
incubated at 37°C for 30–45 min to denature the DNA. After
incubation, the samples were electrophoresed at 1.5 V/cm for 18–20 h
in a 0.7% neutral agarose gel. The DNA in the gel was stained,
acid-depurinated, neutralized, and transferred to a nylon membrane
(40)
. Hybridization experiments were performed in 10 ml of
solution containing 50% (v/v) formamide, 6x SSC, 0.5% SDS, 5%
dextran sulfate, denatured salmon sperm DNA (100 µg/ml), and
1–2 x 108 cpm
32P-labeled single-stranded exon 8-specific
probes generated by PCR (41)
. After hybridization for
20–22 h, the filters were washed to final stringency at 62°C in 1x
SSC/1% SDS and exposed to a phosphorimager screen, and the individual
band intensities were quantitated upon imaging and processing by
Imagequant software (Molecular Dynamics). The rate of removal of
BPDE-DNA adducts within the individual strands of the p53 gene was
determined by quantifying the reappearance of the full-length
restriction fragment bands. The average number of UvrABC-sensitive
sites per fragment was calculated by Poisson distribution equation
(42)
. The filters were also used to expose Kodak X-OMAT
film for autoradiography. The strand-specific repair experiments were
repeated with two independent samples.
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RESULTS |
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. A brief 30-min exposure to doses as low as 0.3
µM anti-BPDE resulted in a
significant increase in p53 protein levels in p53-WT cells. Time course
experiments indicated that the rapid induction response, detectable
within a few hours and peaking at about 8–24 h, was well sustained
beyond a period of 24 h (Fig. 1B)
. A severalfold
increase in the induction of p53 protein was observed with higher doses
of anti-BPDE. Consistent with the wild-type p53 status of
p53-WT cells, the levels of p21, a known transcriptional target of p53,
were also induced severalfold and sustained up to 24 h. Homozygous
p53-Mut cells, harboring a missense Arg
Trp base substitution
mutation at codon 248 of the p53 gene (43)
, showed
constitutively elevated levels of the mutant p53 protein in carcinogen
treated as well as untreated cell populations. No p53 protein was
detectable upon anti-BPDE treatment in homozygous p53-Null
cells containing a frameshift mutation at codon 184 of p53 gene
(43)
. Surprisingly, both the p53-Mut and p53-Null cells
showed a clear induction of p21 protein as a function of
anti-BPDE dose. Time course experiments with 1.2
µM of anti-BPDE demonstrated that
levels of p21 peaked at 8 h in all three cell lines. However, the
induction of p21 was not sustained up to 24 h in the p53-Mut and
p53-Null cells. Nevertheless, it was obvious that anti-BPDE
induction of p21 expression was not dependent upon the presence of
p53-WT activity in human fibroblast cells.
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. For repair experiments, the cells lines were treated with
a dose of 1.2 µM anti-BPDE, which
gave an initial DNA damage level of 40 ± 5
adducts/106 nucleotides. Cells exposed to the
carcinogen for 30 min (0 h) and incubated for increasing periods of
time (up to 24 h) were lysed and analyzed for
anti-BPDE-DNA adducts. Normal human fibroblasts containing
wild-type p53 showed an efficient DNA repair response, removing a
greater percentage of adducts at earlier time points than p53-Mut and
p53-Null cells, which exhibited significantly reduced rates of NER and
overall loss of anti-BPDE-DNA adducts (Fig. 2B)
.
Quantitative comparisons at 24 h after exposure to
anti-BPDE showed that p53-WT cells removed 70% of
anti-BPDE-DNA adducts followed by 50% and 35% by p53-Mut
and p53-Null cells, respectively. A similar differential response in
repair pattern was apparent in the three cell lines at earlier
posttreatment times.
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. The band intensities were quantitated using a Molecular
Dynamic PhosphorImager by analyzing the fragment images left on
phosphor cassettes by the probe hybridized blots. The average number of
UvrABC-sensitive sites per fragment were calculated by the Poisson
distribution equation, and these calculations took into consideration
the UvrABC excinuclease nonspecific cleavage, which was in the range of
0.03–0.16 incisions/16 kb. Under the conditions of our treatment, the
initial frequency of anti-BPDE adducts formed by 2
µM of anti-BPDE was in the range of
1–1.3/16-kb fragment in the DNA of all of the three cell lines. The
normal human fibroblasts containing the wild-type p53 gene efficiently
repaired both TS and NTS of the p53 gene showing 80% repair within
8 h, and the repair was virtually complete within 24 h (Fig. 3B)
. However, slightly reduced initial repair of the NTS
could be seen in p53-WT cells at 4 h posttreatment. Both the
homozygous p53-Mut and p53-Null cells also displayed efficient repair
of TS of the p53 gene, showing repair rates almost comparable to that
of p53-WT cells. Moreover, the repair of the NTS of the p53 gene in
these cell types was considerably less than that observed with p53-WT
cells. Homozygous p53-Mut cells showed about 30 and 40% repair within
8 and 24 h, respectively. Homozygous p53-Null cells exhibited the
least repair, showing about 20% repair of NTS within 24 h.
Consistent with their inefficient capacity for GGR, these results
indicated that the p53-deficient cells lacked the ability to repair
chemical carcinogen-induced DNA adducts from NTS while their TCR
activity remained unaffected by the absence of p53 protein.
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. The cells lacking p53 protein (p53-Null) exhibited lowest
survival, indicating greater reproductive cell death. The short-term
viability assessments of cell lines were in concordance with the
results of survival assays. The cell lines exposed to 1.2
µM of anti-BPDE showed significant
differences in carcinogen cytotoxicity at 24, 48, and 72 h (Fig. 4B)
. Although all of the three cell lines exhibited a
carcinogen-induced decrease in the extent of dye metabolizing
functional cell population, the p53-WT cells were relatively more
resistant than p53-Mut and p53-Null cells. The differences in the
cytotoxic response, as indicated by cell viability, was more pronounced
when cells were analyzed after 72 h than at earlier time points.
The homozygous p53-Null cells showed the highest cytotoxicity in
comparison to p53-Mut and p53-WT cells. In addition, there was a
significant recovery in viable p53-WT cells upon maintaining in growth
medium for 72 h after chemical carcinogen exposure.
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. On the other hand, p53-Mut and p53-Null cells
demonstrated significant DNA fragmentation, and the size of DNA
fragments was greatly reduced, reaching the expected 180-bp nucleosome
monomer size for the p53-Null cells at 48 h. Quantitative
assessment of apoptotic index in the treated cell populations indicated
a gradual increase in the released DNA (apoptotic fraction) as the
incubation time of cells was increased. The level of DNA fragmentation
was 15% for p53-WT cells followed by 30% for p53-Mut cells (Fig. 5B)
. The highest level of apoptosis was seen with p53-Null
cells reaching up to 43% at 48 h, and these cells also showed a
rapid DNA fragmentation response after anti-BPDE exposure.
Furthermore, cytological examination of fluorescent dye-stained p53-Mut
and p53-Null cells also revealed morphological changes typical of
apoptosis i.e., DNA condensation, membrane blebbing, cell
shrinkage, and apoptotic bodies. No such changes were prominent in the
population of treated p53-WT cells.
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DISCUSSION |
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We have previously reported that p53-Mut and p53-Null cells are deficient in GGR as well as the repair of UV-induced pyrimidine dimers from the NTS compared to cells having a functional wild-type p53. To extend these findings to chemical genotoxin, the present study used (±)-anti-BPDE as the model DNA-damaging agent. Demonstration of an association between anti-BPDE-induced DNA adducts and an increase in wild-type p53 protein suggested a direct cause-effect relationship between anti-BPDE-DNA damage and p53 response. Constitutive high level expression of mutant p53 protein was detected in p53-Mut cells, and a complete absence of p53 protein in p53-Null cells was seen irrespective of the level of anti-BPDE-induced DNA damage. In addition, our results indicate that wild-type p53 is not an absolute requirement for the expression of p21, implicating the presence of additional pathways. This is further strengthened by the fact that the p21 induction response was qualitatively comparable in all of the three cell lines despite the presence or absence of wild-type p53 function. Although initial reports have shown that the p21 protein induction is p53-mediated (52 , 53) , recent studies with p53 null and LFS cells have shown that p53 is dispensable for p21 induction mediated by UV exposure and serum-starvation (46 , 47) . Furthermore, the now well established ability of the p53 homologue, p73, to induce p21 and apoptosis also suggests the presence of additional p53-independent mechanisms (54 , 55) .
Several investigations based upon assessing the effect of p53-WT on the
efficiency of repairing genotoxic damage have suggested a direct
participation of p53 protein in the damage excision process. Transient
transfection of RKO cells containing wild-type p53 function, with
vectors expressing genes that abrogate wild-type p53 activity, resulted
in decreased host-cell reactivation of exogenously UV-irradiated
reporter plasmid and NER (56)
. Decreased repair in the
absence of wild-type p53 function has also been reported by directly
measuring the repair of UV-induced lesions in LFS fibroblasts of
different p53 status (16)
. The results presented here are
in agreement with these studies that show a significant difference in
NER rates of anti-BPDE-induced bulky DNA adducts in human
LFS fibroblasts. Besides, all of the three human fibroblasts maintained
the preferential and rapid repair within TS of the p53 gene
irrespective of p53 status. However, the repair of the NTS was faster
and virtually complete within 24 h in p53-WT cells in comparison
to reduced repair in p53-Mut cells. No such enhancement in repair was
apparent within the NTS of the p53 gene in p53-Null cells even at
24 h (Fig. 3)
. These results suggest that p53 may not be a
necessary component of the active TCR complex, but may be involved in
global repair that is also responsible for the lesion elimination
within NTS. Our results are further strengthened by the fact that p53
can interact with the repair factors, ERCC-3 and p62, present in
transcription factor IIH, which is essential for NER and
transcription initiation (18
, 37
, 43
, 50)
. The
intermediate levels of repair seen in p53-Mut cells suggest that the
mutant p53 protein with codon-248 mutation might retain partial
function. X-ray crystallography studies have shown that the Arg at
codon 248 present in the central domain of the protein is critical for
p53 protein-DNA contacts and in turn the transcriptional activation
function of p53 (57)
. However, functional studies indicate
that this mutation does not confer a dominant negative function of the
mutant protein (over the p53-WT protein) like other mutations seen in
Li-Fraumeni patients (58
, 59)
. In addition,
protein-protein interactions of p53 with the transcriptional complex
and various repair proteins map to the NH2
terminus and the COOH terminus of the protein, giving rise to the
possibility that the mutant protein may be active, albeit
inefficiently, for the repair function(s) of p53. Thus, the enhanced
DNA repair of p53-Mut cells over that of p53-Null cells could very well
be attributed to the maintenance of p53-repair protein interactions.
Because inefficient repair of NTS has been implicated in mutagenesis
and carcinogenesis, one would expect an increase in the mutation
frequency in the absence of p53. However, a similar mutation frequency
of the lacI transgene in p53 wild type as well as deficient
backgrounds has been reported in an earlier study (60)
.
Although the above mentioned study had used a different model system
and determined different end points of DNA damage (DNA repair rate
versus mutations), additional studies are required to
dissect the complete role of p53 in the molecular mechanisms of DNA
repair and biological consequences of altered p53 function.
In conjunction with the effect of p53 on NER, we also observed that the p53-Null and p53-Mut cells were more sensitive to the cytotoxic effects of anti-BPDE-induced DNA lesions than the cells expressing wild-type p53. Although the p53-WT cells showed a dose-dependent decrease in clonogenic survival and viability, they were relatively more resistant and able to promptly recover under full growth conditions. The cells with nonfunctional p53 do not seem to possess a capacity to recover and produce a higher fraction of cells in the survival assays. The ability of p53-WT cells to show such a recovery might be attributed to their capacity for the efficient removal of potentially cytotoxic lesions from their genome, and it could be argued that the presence of p53-WT function confers anti-BPDE resistance through a DNA repair related mechanism. It is well documented that the relatively high cytotoxic and mutagenic potential of anti-BPDE adducts results from their slower repair as well as their interference on DNA replication and transcription (61) . In addition, this cellular recovery phenomenon exhibited by normal human fibroblasts could very well depend on proficient DNA excision repair because NER deficient XP cells are incapable of such recovery (62 , 63) .
Presence of wild-type p53 also enables cells to arrest at the
G1-S stages of the cell cycle and undergo
apoptotic cell death after DNA damage. The apoptotic response is
generally cell-type-dependent, and human fibroblast cells are known to
show resistance to apoptosis upon genotoxic insult (36
, 64
, 65)
. In addition, p53-independent apoptotic pathways have been
observed in normal proliferating lymphoid cells (66)
and
in a variety of other tumor cell types (67)
. The data
presented here are in agreement with a clear lack of apoptosis in
normal human fibroblast cells containing wild-type p53. On the
contrary, cells with mutant p53 or no p53 protein readily and rapidly
undergo apoptosis after genotoxic exposure. The induction of apoptosis
recognized by DNA fragmentation was apparent within 4–6 h (Fig. 5A)
. Quantification of apoptotic process demonstrated that
in contrast to p53-Mut and P53-Null cells, the p53-WT cells are
resistant to the induction of apoptosis (Fig. 5B)
. The
results also indicate that anti-BPDE-induced damage invokes
Phase I fragmentation, resulting in an early release of high molecular
weight DNA in both the cell types (36
, 68)
. The data
presented here are consistent with our previous findings and with
recent reports, whereas wild-type p53 function abrogated by HPV E6 in
human fibroblast cells resulted in decreased GGR, enhanced cytotoxicity
and apoptosis upon UV-irradiation (69
, 70)
. Based on the
overall data and our previous findings, we suggest that the persistent
cytotoxic lesions within the NTS that escape repair are responsible for
the induction of apoptosis and cytotoxicity seen in p53-deficient
cells.
In summary, the data show that the loss of p53-WT function is associated with a decrease in the repair of genotoxic lesions within overall genome and the inefficient removal of anti-BPDE-induced DNA lesions from NTS without any significant effect on TS support a clear role of p53 tumor suppressor gene in GGR. Although the mechanism by which p53 activity affects GGR remains unknown, our results demonstrate that cell death can be a p53-independent response, but dependent on DNA damage of the genome that evades repair. Further unraveling of damage response, cell cycle, and the apoptotic pathway and identification of key determinants of eukaryotic repair complex is expected to lead to more innovative approaches for understanding the intricacies of DNA damage processing at various levels of genomic organization.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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1 Supported by National Institute of Environmental
Health Sciences Grant ES2388. 
2 Present address: Division of Molecular Virology,
Baylor College of Medicine, Houston, TX 77030.
3 To whom requests for reprints should be
addressed, at 103 Wiseman Hall, 400 W. 12th Avenue, Columbus, OH 43210.
Phone: (614) 292-9375; Fax: (614) 292-7237; E-mail: wani.2{at}osu.edu
4 The abbreviations used are: NER, nucleotide
excision repair; BPDE,
benzo(a)pyrene-diol-epoxide; GGR, global genomic
repair; LFS, Li-Fraumeni syndrome; MTT, methylthiozole tetrazolium;
NTS, nontranscribed strand; p53-Null, p53-nullizygous; p53-Mut,
p53-mutant; p53-WT, p53-wild type; TCR, transcription-coupled repair;
TS, transcribed strand.
Received 11/18/99. Accepted 2/17/00.
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D. P. Miller, G. Liu, I. De Vivo, T. J. Lynch, J. C. Wain, L. Su, and D. C. Christiani Combinations of the Variant Genotypes of GSTP1, GSTM1, and p53 Are Associated with an Increased Lung Cancer Risk Cancer Res., May 1, 2002; 62(10): 2819 - 2823. [Abstract] [Full Text] [PDF]
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J. L. Torino, M. S. Burger, C. A. Reznikoff, and S. Swaminathan Role of TP53 in repair of N-(deoxyguanosin-8-yl)-4-aminobiphenyl adducts in human transitional cell carcinoma of the urinary bladder Carcinogenesis, January 1, 2001; 22(1): 147 - 154. [Abstract] [Full Text] [PDF]
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 P.C. HANAWALT, D.J. CROWLEY, J.M. FORD, A.K. GANESAN, D.R. LLOYD, T. NOUSPIKEL, C.A. SMITH, G. SPIVAK, and S. TORNALETTI Regulation of Nucleotide Excision Repair in Bacteria and Mammalian Cells Cold Spring Harb Symp Quant Biol, January 1, 2000; 65(0): 183 - 192. [Abstract] [PDF]
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M.-C. Mathieu, I. Lapierre, K. Brault, and M. Raymond Aromatic Hydrocarbon Receptor (AhR){middle dot}AhR Nuclear Translocator- and p53-mediated Induction of the Murine Multidrug Resistance mdr1 Gene by 3-Methylcholanthrene and Benzo(a)pyrene in Hepatoma Cells J. Biol. Chem., February 9, 2001; 276(7): 4819 - 4827. [Abstract] [Full Text] [PDF]
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