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Experimental Therapeutics |
Department of Radiation Oncology, Massey Cancer Center, Medical College of Virginia, Virginia Commonwealth University, Richmond, Virginia 23298-0058
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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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Although several reports have shown that XPA cells can be rescued by increased UV resistance or increased levels of HCR (HCR) by cell transfection with the XPA gene or the phage T4 denV gene (3, 4, 5, 6, 7, 8) , to date there has been no demonstration of sensitizing tumor cells proficient in NER with a vector expressing a protein that interferes with NER as a strategy for cancer therapy. The XPA protein is a zinc-finger protein of Mr 31,000 (273 amino acids) involved in DNA damage recognition (4 , 9 , 10) . Previous work demonstrated that the XPA protein interacts with the ERCC1 protein through amino acid residues 72–84 of XPA (11, 12, 13) . Hence, we argued that by overexpressing a truncated form of the XPA protein encompassing this domain, one might be able to prevent the ERCC1/XPF nuclease complex from associating with the normal XPA protein produced by the cell and would thus interfere with the early stages of NER. By taking advantage of the efficient gene delivery and protein expression properties of adenovirus (3 , 14) , the XPA protein is an excellent target for gene therapeutic intervention through increased sensitization of tumor cells to chemotherapy. This strategy is particularly interesting when applied to tumor cells, allowing for more efficient therapies with a variety of drugs used for chemotherapy and to counteract increased resistance to chemotherapeutics frequently seen during the course of treatment of certain types of cancers. We demonstrate in this report that infection of human tumor cells proficient in NER with a replication-defective adenovirus expressing amino acids 59–114 of the XPA protein sensitizes the cells to UV and cisplatin.
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MATERIALS AND METHODS |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Construction of Adenovirus-expressing Flag-tagged XPA Proteins.
A truncated version of the human XPA cDNA with a 97-bp deletion between
position 104 and 200 (4
, 20)
was cloned in the correct
reading frame in pCMV-FLAG-2 (Kodak, Rochester, NY) and was expected to
produce a FlagXPA fusion protein consisting of amino acids 59–273 of
the human XPA protein (4)
. We previously demonstrated that
SV40-transformed human XPA fibroblasts stably transfected with a
plasmid expressing this portion of the XPA gene show normal
HCR of a UV-damaged reporter gene (17)
. Others have also
noted that amino acids 1–58 of XPA are not required for conferring UV
resistance (11
, 21)
. The
FlagXPA59–273 DNA cartridge was transferred into
pZeroTG-CMV and made into an adenovirus (14)
.
Subsequently, a 3' deletion was made of the XPA cDNA in
pZeroTG-CMVFlagXPA59–273 by removing the DNA
sequence 3' of the unique BsaBI site (4)
. This
plasmid is expected to produce a truncated XPA protein of 55 amino
acids (59–114) tagged at its NH2 terminus with
Flag and spanning the ERCC1 binding domain (amino acids 72–84).
Recombinant viruses were screened by Western blotting of extracts
obtained from infected 293 cells using anti-Flag antibody. Viral clones
that produced proteins of the expected sizes that reacted with the
anti-Flag and anti-XPA antibodies (20)
were used in
subsequent experiments. Adenoviruses expressing ß-galactosidase
(AdLacZ), GFP (AdCMV-GFP), Flagp38 MAP kinase
(AdCMV-Flagp38)4
under control of the CMV promoter or an "empty" adenovirus
(AdCMV) without any trans-gene, were constructed and used in
HCR assays or as virus controls.
Protein Purification and Immunoblotting.
For Western blot analyses, A549 or 293 cells were infected with viruses
expressing XPA proteins or various control viruses and harvested 2–3
days postinfection. Samples were then briefly sonicated in loading
buffer and boiled prior to SDS-PAGE and Western analysis. For affinity
purification of FlagXPA proteins, anti-Flag beads (Kodak, Rochester,
NY) were incubated with protein extracts isolated from cell monolayers
by washing cells with PBS, then lysing the cells for 30 min on ice in
buffer consisting of 50 mM Tris-HCl, 0.25 M
NaCl, 0.1% NP40, 5 mM EDTA, 1 mM DTT and
protease and phosphatase inhibitors (protease inhibitor mixture
and phosphatase inhibitor mixture 1 and 2; Sigma Chemical Co.,
St. Louis, MO) at the manufacturer’s recommended dilutions. After
centrifugation, the supernatant was transferred to a separate tube, and
the cell pellet was reextracted using the same lysis solution twice
more for 10 min on ice. The pooled supernatants were then bound to
anti-Flag affinity beads in solution for 3 h at 4°C. After
centrifugation, the supernatant was discarded, and the beads were
washed twice with lysis buffer. After washing, the beads were
resuspended in a small volume of 10 mM TE buffer [Tris-HCl
and 1 mM EDTA (pH 8.0)], and 10 µg of Flag
peptide (Kodak, Rochester, NY) was added to release bound FlagXPA
protein complexes from the beads. The reaction was incubated on ice for
30 min and then centrifuged. The supernatant containing the eluted
proteins was transferred to a new tube and used for analysis by Western
blotting. Protein samples were typically separated on a 4–20%
Tris-Gly gel (Novex, San Diego, CA) at 125 V for 2 h and then
transferred to a polyvinylidene difluoride membrane for 2 h
at 0.6 A. After blocking in 5% milk buffer, the membranes were
incubated with either mouse anti-Flag antibody (Kodak, Rochester, NY),
mouse anti-ERCC1 antibody (Neomarkers, Freeman, CA), or polyclonal
rabbit XPA antibody (20)
. Antibody binding was detected
with a secondary IgG antibody conjugated with alkaline phosphatase
(Santa Cruz, Santa Cruz, CA), followed by detection by
chemiluminescence (Tropix, Bedford, MA). Cellular XPA appears as a band
of Mr 
40 on a gel.
DNA Repair Assays.
A modification of our earlier procedure was followed for HCR assay
(3)
. Briefly, a confluent cell monolayer in a 6-cm tissue
culture dish was infected with adenovirus (XPA virus or control virus)
at a MOI of 3–30 and after a 3 h incubation at 37°C
while slowly rocking, the medium was removed and replaced with fresh
culture medium. After 2 days, infected cells were trypsinized, and
reseeded in triplicate in a 96-well microtiter plate as 2-fold serially
dilutions with 1 x 105 cells
seeded in the first row. On the following day, AdLacZ irradiated with
increasing doses of UV (0–400 J/m2) was added to
the 96-well plate at a MOI of 10 for 3 h, then removed, and
replaced with fresh media. After 3 days, the medium was removed from
all of the wells, and the cells were fixed with a solution of 3%
paraformaldehyde/0.25% glutaraldehyde for 10 min at room temperature.
After discarding the fixative and washing the plate with PBS, 50 µl
of the fluorescent substrate resorufin-ß-d-galactose
(Boehringer-Mannheim, Indianapolis, IN) at 0.15
mM in PBS supplemented with 1
mM MgCl2 was added to each
well, and the plate incubated at room temperature in the dark for
1 h. The plate was then read on a FluoroCount (Packard, Meriden,
CT) fluorometer using an excitation filter of 530 nm and an emission
filter of 620 nm. Fluorescence readings from cells not infected with
AdLacZ were subtracted from all other readings as background. Relative
HCR was determined by dividing the average readings for a given UV dose
by the average readings from wells with cells infected with undamaged
AdLacZ. A larger relative fluorescence indicates larger expression of
ß-galactosidase and more DNA repair. GFP fluorescence of cells
infected with AdCMV-GFP is negligible at these filter settings.
To determine the removal of UV damage, a modification of the immune assay for pyrimidine dimers was followed (22, 23, 24) . Briefly, 75% confluent dishes of A549 cells were infected with AdCMV, AdCMV-FlagXPA59–273, or AdCMV-FlagXPA59–114 at a MOI of 10. Two days after infection, cells were trypsinized and reseeded at 1:8 in 6-cm dishes. One dish was used for each experimental point including a dish for unirradiated control. The next morning, dishes were UV irradiated at a dose rate of 2 W/m2 to a total dose of 5 or 10 J/m2. The cells were then harvested at various times and cell pellets frozen on dry ice. Cell lysis buffer [0.1 M Tris (pH 7.5), 0.15 M NaCl, 12.5 mM EDTA, and 1% SDS) containing 200 µg/ml proteinase K was added to the cell pellets and sonicated followed by incubation at 42°C for 5 h. DNA was isolated by phenol:chloroform extraction followed by ethanol precipitation. The DNA was resuspended in 200 µl of TE buffer with 5 µg of RNase A and incubated at 37°C for 2 h, followed by a second phenol:chloroform extraction and ethanol precipitation. The DNA was resuspended in 10 µl of TE, and 1 µl was spotted in triplicate on duplicate GeneScreenPlus (NEN) membranes. When dry, the membranes were blocked with 5% nonfat dry milk in TBS-T [0.1 M Tris-HCl (pH 7.5), 0.1 M NaCl, and 0.005% Tween 20] overnight at 4°C. The membranes were then washed briefly in TBS-T, and incubated with a 1:3000 dilution of either anti-CBPD or anti-PP (Kamiya Biomedical, Seattle, WA) mouse monoclonal antibody in 5% BSA in TBS-T for 1 h at room temperature. After washing with TBS-T, the membranes were incubated with a 1:3000 dilution of antimouse alkaline phosphatase (Santa Cruz Biotechnology, Santa Cruz, CA) in TBS-T for 1 h and washed several times in TBS-T followed by incubation with CDP-Star (NEN), and exposure to a chemiluminescence screen. Spots were quantified using volume analysis on a Bio-Rad Molecular Imager FX using Quantity One software (Bio-Rad, Hercules, CA). To normalize the amount of DNA spotted, the membranes were hybridized to 32P-labeled genomic DNA isolated from A549 cells. Briefly, membranes were prehybridized in Church buffer [1% BSA (w/v), 7% SDS (w/v), 0.25 M phosphate buffer (pH 7), and 1 mM EDTA] containing salmon sperm DNA (0.1 mg/ml) for blocking for several hours at 65°C. 32P-labeled genomic DNA labeled with a "Prime-it" kit (Stratagene, La Jolla, CA) was then added to fresh prewarmed Church buffer and hybridized overnight at 65°C. Membrane were washed in Church wash buffer [1% SDS in 40 mM phosphate buffer (pH 7.0) and 0.5 mM EDTA]. Membranes were exposed to a phosphorimaging screen, scanned in Molecular Imager, and spots quantified using volume analysis. To analyze the data, the signal from the immunoassay was normalized to the DNA bound to the membrane. The signal from the spots obtained without any UV irradiation was subtracted from all of the values to adjust for background levels. Finally, to obtain the fraction of damage remaining, the normalized, background-adjusted signals obtained from the immunoassay were divided by the mean value for the 0 min time points.
Cytotoxicity Assay.
Confluent 6-cm dishes of A549, GM637, and XP12RO(M1) cells were
infected with a MOI of 3 with either
AdCMV-FlagXPA59–114,
AdCMV-FlagXPA59–273, or control virus. For the
UV toxicity assay, infected cells were incubated for 48 h, then
trypsinized and UV irradiated in a small droplet of PBS at a dose rate
of 1 W/m2. Treated cells were plated in
triplicate in 96-well plates and serially diluted. For the cisplatin
toxicity assay, infected cells were seeded in 96-well plates at
different dilutions at 24 h, allowed to attach overnight, and then
treated with cisplatin (Aldrich, St. Louis, MO) the following day for
4 h at 37°C in complete media. After either treatment, the
plates were incubated for 5 additional days. Finally, Resazuri
(AlamarBlue; Molecular Probes, Eugene, OR) was added to the medium to a
final concentration of 10 µM, and the plates were
incubated for 3 h at 37°C and then read in the FluoroCount
reader at an excitation of 530 nm and an emission of 590 nm. A larger
relative fluorescence signal indicates more live cells and, thus, lower
toxicity. The results were corrected and normalized as described above
for the HCR assay.
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RESULTS |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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To demonstrate expression of the FlagXPA proteins from the constructed
adenoviruses, A549 cells were infected with either
AdCMV-FlagXPA59–114 or an adenovirus producing
functional XPA protein, AdCMV-FlagXPA59–273, at
an MOI of 30. Although, the XPA protein is
Mr 31,000, it migrates as
Mr 40,000 by SDS-PAGE
(9)
. After 3 days of incubation, the cells were harvested
and processed for SDS-PAGE. After protein separation on the gel,
proteins were transferred to a membrane and probed with anti-Flag
antibody for detection of the FlagXPA proteins (Fig. 1A)
. Infection of cells with
AdCMV-FlagXPA59–273 expressing near full-length
Flag-XPA59–273 protein gave a band of
Mr 40,000, as expected, and
infection with AdCMV-FlagXPA59–114 produced a
single band of less than Mr 5,000 as
would be expected for this construct. Termination of the truncated XPA
protein is expected to occur in a stop codon residing in the flanking
sequence. To unequivocally demonstrate that these Flag proteins were
indeed of XPA origin and expressed from the viruses, we then performed
parallel immunoblots using either anti-Flag or anti-XPA antibody (Fig. 1B)
. We found that both of the FlagXPA proteins also reacted
with the anti-XPA antibody. We occasionally detect a second XPA band by
Western analysis at a position of Mr
60,000 (Fig. 1B
, *), which disappears when the sample is
diluted. It is presently unknown why this happens, but is possible that
the FlagXPA59–273 protein is not entirely
dissociated from other proteins or else forms dimers at higher
concentrations. Alternatively, the top band could be attributable to
translational read-through or to different conformational properties of
XPA resulting in aberrant migration by SDS-PAGE (9)
. In
any case, this result demonstrates the efficient expression of FlagXPA
proteins from adenovirus in A549 cells.
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, left). It is clear from the result shown that ERCC1 binds
specifically to the FlagXPA59–114 protein
because a band of Mr 40,000, which
is the relative molecular weight of ERCC1 (26)
, is
detected only in extracts from cells infected with the
AdCMV-FlagXPA59–114. No such band appears in the
lane with an extract from cells infected with the AdCMV-Flagp38 control
virus. The same membrane was then reprobed with anti-Flag antibody to
ensure the presence of Flag proteins in the extracts (Fig. 2
,
right). Indeed, Flag proteins of the expected sizes,
i.e., Mr 5,000 for
FlagXPA59–114 and 38,000 for Flagp38 MAP kinase,
were found in the affinity purified complexes. This result demonstrates
that ERCC1 specifically interacts with
FlagXPA59–114 in vivo.
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. Again, A549 cells were infected with different MOI
(3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30)
. In time course experiments, expression of FlagXPA proteins was
detected on day 1, and then increased on days 2 and 3 (data not shown).
On day 4, FlagXPA levels were roughly equivalent to those on day 3. We
found that the AdCMV-GFP and the
AdCMV-FlagXPA59–273 viruses produced no
significant increase in cell toxicity even at the highest MOI of 30.
The relative growth of cells infected with AdCMV-GFP compared with
uninfected cells was 0.94, and with
AdCMV-FlagXPA59–273, it was 1.03. However,
infection of A549 cells with AdCMV-FlagXPA59–114
did prove toxic to cells, resulting in a relative growth of 0.56 at a
MOI of 30. In parallel, samples were analyzed for the expression of FlagXPA59–114 (Fig. 3B)
. An
excellent correlation between cell toxicity/growth inhibition and
expression of FlagXPA59–114 was noticed.
With a MOI of 3 that produced only minor toxicity, no
FlagXPA59–114 protein was detected by Western
analysis, whereas at higher MOI, both toxicity and expression were
noted. This result demonstrates that
FlagXPA59–114 protein is toxic to cells when
expressed at increased levels, which suggests that XPA or ERCC1
function is required even without any exogenously inflicted DNA damage
to the cell. In subsequent experiments, we used a low MOI because of
the relative toxicity of AdCMV-FlagXPA59–114 in
these cells.
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. HCR is an indirect assay for DNA repair that relies on the notion
that damage needs to be removed in order for transcription to occur,
and, thus, the greater the relative reporter gene activity, the more
proficient the DNA repair (3
, 7)
. At a dose of 400
J/m2, the ß-galactosidase activity in cells
that had not been infected with virus prior to HCR was 0.72 relative to
readings from cells infected with undamaged AdLacZ. By comparison, the
ß-galactosidase activity in control cells initially infected with
AdCMV-GFP and then with UV-irradiated AdLacZ was 0.94. At the same UV
dose, cells infected with AdCMV-FlagXPA59–114
showed a significantly reduced fraction of ß-galactosidase activity
of 0.29 (P = 0.0057). This result
demonstrates that infection of A549 cells with
AdCMV-FlagXPA59–114 impairs HCR and also
suggests that perhaps cell survival after UV would be reduced.
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. Growth of uninfected cells exposed to 45 J/m2
of UV was reduced to 0.70 compared with cells not exposed to UV.
Similarly, cells exposed to the same UV dose after infection with the
control virus AdCMV reduced growth to 0.60. However, cells infected
with AdCMV-FlagXPA59–114 at a MOI of 3 before
exposure to UV showed a significantly greater reduction in growth of
0.47 (P = 0.0387). Because UV irradiation of
cells were carried out in a droplet, relatively high UV doses were
required to see toxicity under these experimental conditions. To
confirm this result using other cells and to establish the relative
effect of AdCMV-FlagXPA59–114 infection on the
UV sensitivity of repair-competent cells to that of XPA cells, a second
experiment was carried out with NER-deficient XPA [XP12RO(M1)] and
normal NER-proficient (GM637) fibroblasts (Fig. 6)
. As expected, both uninfected and control-infected XP12RO(M1) cells
were much more sensitive to UV than were GM637 cells, and
AdCMV-FlagXPA59–273 expressing nearly
full-length XPA protein rescued XP12RO(M1) cells to GM637 levels. Most
importantly, infection of GM637 with
AdCMV-FlagXPA59–114 resulted in UV sensitization
that approached the XP12RO(M1) levels. These results demonstrate that
infection of two different human cell lines that are normal in NER with
AdCMV-FlagXPA59–114 UV sensitizes the cells to
levels approaching those of XPA.
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. Uninfected cells given a dose of 20 µM cisplatin for
4 h showed reduced growth compared with untreated cells of 0.49,
and cells infected with AdCMV control virus before the same cisplatin
treatment were slightly more sensitive with a fraction of growth
relative to untreated infected cells of 0.32. Cells infected with
AdCMV-FlagXPA59–114 before challenge with
cisplatin were much more sensitive, showing only 0.15 of the growth of
cells infected with the same virus but not exposed to cisplatin
(P = 0.0026). These results demonstrate that
infection with AdCMV-FlagXPA59–114 renders cells
more sensitive to cisplatin as well as to UV, which suggests
that this approach could potentially be used to increase the
sensitivity of tumor cells to cisplatin and other clinically important
drugs that produce bulky DNA damage.
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. As expected, we found that PP lesions were removed much
faster from the DNA than were CBPDs with half lives of <1 h and
15–24 h, respectively, in agreement with previous reports (22
, 24
, 27) . We found no difference in the removal of CBPDs between
uninfected cells, cells infected with AdCMV (control), and cells
infected with AdCMV-FlagXPA59–114 (Fig. 8B)
, which suggests that expression of
FlagXPA59–114 did not interfere with the removal
of CBPDs. However, when the removal of PPs were examined, we found
dramatic differences between cells infected with
AdCMV-FlagXPA59–114 compared with uninfected and
AdCMV-infected cells (Fig. 8C)
. To our surprise, cells
infected with AdCMV-FlagXPA59–114 appeared to
generate more PPs over time compared with controls. Because it is
inconceivable that more lesions were generated with time after UV
irradiation, the most reasonable interpretation of this result is that
the PP epitope is better exposed to the antibody, perhaps resulting
from partial DNA repair of the PPs. In any case, the results suggest
that the removal of PPs but not CBPDs is affected by the expression of
FlagXPA59–114.
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DISCUSSION |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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We previously reported on a virus similar to the one described here that expressed the same truncated XPA protein without the Flag epitope that sensitized cells to UV (33) . However, we were unable to clearly show expression of this truncated protein in infected cells. Detecting the presence of the Flag epitope on the truncated XPA protein made by the virus described in the present study unequivocally demonstrates that this protein is made by the virus and not by the cell. Results from the present study confirm earlier findings that ERCC1 binds to XPA (12 , 13) . However, the in vivo binding between full-length ERCC1 and XPA proteins was previously demonstrated only with GAL4-fusion proteins in a yeast two-hybrid screen (11 , 13) . The result from our study clearly demonstrates that interaction between the 59–114-amino-acid domain of XPA and ERCC1 occurs in human cells in vivo. We are currently investigating whether other proteins, including XPF (12) , are also present in this protein complex. The presence of an epitope tag combined with the efficient expression from a highly infectious adenovirus should be very useful in isolating large quantities of XPA protein complexes and for determining the composition and changes in posttranslational modifications in these complexes that occur in vivo in response to DNA damage.
It was demonstrated previously that a mutant XPA (
G) with 4 amino
acids (GGGF) deleted in the G-domain was unable to bind ERCC1 but was
able to compete with normal XPA protein and to reduce DNA repair
in vitro (11)
. It was suggested that G
reduced repair because its DNA binding ability was still retained but
was not loading ERCC1 protein on to the damaged DNA site
(11)
. Our results suggest that the reciprocal event is
also possible, i.e., the
FlagXPA59–114 protein encompassing the G-domain
is able to bind ERCC1, and perhaps the resulting complex competes with
the formation of normal XPA/ERRC1/(XPF?) complexes. However, at this
point, we have no direct evidence that this occurs. Very recently, it
was shown that the interaction between XPA and ERCC1 is modulated
indirectly by a protein kinase (34)
, which would provide
another means by which NER may be inhibited to increase the
sensitization of tumor cells to chemotherapy.
The results from this study demonstrate that overexpressing truncated XPA protein is toxic to cells even without treating the infected cells with any DNA-damaging agent, which suggests that either NER is needed at all times or that the function of some other protein needed for normal cellular growth is inhibited by the truncated XPA protein. However, we were able to establish the virus dose range within which a sensitizing effect on treatment with UV or cisplatin could be clearly distinguished from the toxicity observed from the virus itself. Perhaps by controlling the expression of the FlagXPA59–114 protein by using an inducible promoter, one could alleviate the toxicity of the protein when cells are not treated with a drug. Along the same line, future virus vectors may be designed with a DNA damage-inducible promoter to reduce the expression of truncated XPA in the absence of DNA damage and, thus, limit toxicity in the absence of drug treatment. Use of such vector would be expected to improve the therapeutic ratio.
How might the expression of Flag-XPA59–114 affect NER? From these studies, we can conclude that the most likely mechanism for the increased cell toxicity of AdCMV-FlagXPA59–114 is that expression of FlagXPA59–114 interferes with the removal of PP lesions. This effect is specific for PPs because the removal of CBPDs was not affected. Of interest is the result from the immune assay that suggests that the PPs in the DNA isolated from cells infected with AdCMV-FlagXPA59–114 is exposed more efficiently to the antibody specific for the PP epitope. The most reasonable explanation for this result is that the PP has been altered in some fashion but is still attached to the DNA. Conceivably, this alteration could be attributable to the incision on one side of the lesion, presumably the 3' side (35) , resulting in a ‘flap’ that is more exposed to the antibody and thus would result in an increased signal. However, at the present time, we can only speculate as to what the exact structure of this lesion might be.
In summary, our results suggest that using an adenoviral vector expressing a truncated XPA protein to inhibit NER in tumor cells could be an effective combined modality for the treatment of various cancers with drugs known to cause bulky lesions repaired by NER. With this approach, the targeting of tumor cells should be more specific, and perhaps the doses of chemotherapy could be lowered, resulting in more effective treatment with fewer side effects to the patient.
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FOOTNOTES |
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1 This work was supported by NIH PHS
CA53119. 
2 To whom requests for reprints should be
addressed, at Department of Radiation Oncology, Massey Cancer Center,
Medical College of Virginia, Virginia Commonwealth University, P. O.
Box 980058, Richmond, Virginia 23298-0058. Phone: (804) 628-1004; Fax:
(804) 828-6042; E-mail: KVALERIE{at}HSC.VCU.EDU
3 The abbreviations used are: XP,
xeroderma pigmentosum; XPA, XP complementation group A; CBPD,
cyclobutane pyrimidine dimer; ERRC1, excision repair
cross-complementing protein 1; GFP, green fluorescent protein; HCR,
host-cell reactivation; MOI, multiplicity/multiplicities of infection;
NER, nucleotide excision repair; PP, 6-4 photoproduct; TBS-T,
Tris-buffered saline with Tween 20; MAP, mitogen-activated protein;
CMV, cytomegalovirus.
Received 5/15/00. Accepted 11/13/00.
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REFERENCES |
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), or
AdCMV-FlagXPA59–273 (
). B, Western blot
of FlagXPA59–114 protein with increasing MOI in A549
cells.
); cells infected with AdCMV
(
), cells infected with AdCMV-FlagXPA59–273 (
,
), or cells infected with AdCMV-FlagXPA59–114 (
). Closed symbols, data from GM637A cells;
open symbols, data from XP12RO(M1) cells. There is
a statistically significant difference between the cytotoxicity of
GM637 cells infected with AdCMV and that with
AdCMV-FlagXPA59–114 at 45 J/m2
(P = 0.0023) using an unpaired
t test. This experiment was carried out two times with
similar result.
