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Lady Davis Institute for Medical Research, Sir Mortimer B. Davis Jewish General Hospital, Departments of Medicine and Oncology [J. B., N. B., A. E. K., M. A. A-J.], Pharmacology and Therapeutics [M. A. A-J.], Division of Experimental Medicine [P. J. S., A. E. K., M. A. A-J.], and McGill Centre for Translational Research in Cancer [M. A. A-J.], McGill University, Montreal, Quebec, H3T 1E2 Canada
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ABSTRACT |
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 Top ABSTRACT IntroductionMaterials and MethodsResultsDiscussionREFERENCES |
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B-kinase. Here we demonstrate
that PKR is involved in the cellular response to genotoxic stress.
PKR-deficient mouse-embryonic fibroblasts
(PKR-/-) are hypersensitive to bulky
adduct DNA damage caused by cisplatin, melphalan, and UV radiation but
not to other DNA-damaging agents such as Adriamycin. PKR-deficient
cells are highly susceptible to cisplatin-induced apoptosis. They
demonstrate retarded cisplatin adduct removal kinetics. Most
strikingly, PKR localizes to the nucleus rapidly upon cisplatin
treatment. Restoration of PKR in PKR-/-
cells results in resistance to cisplatin and enhanced cell capacity to
remove cisplatin DNA adducts. We conclude that PKR has a function in
the regulation of cellular response to bulky adduct-inducing agents,
possibly by modulating DNA repair mechanisms. |
Introduction |
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TopABSTRACTIntroductionMaterials and MethodsResultsDiscussionREFERENCES |
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PKR is involved in a number of signaling pathways and is emerging as a
mediator of several stress response, antiproliferative, and
apoptotic programs (reviewed in Ref. 8
). Although its
classical function in virally-induced apoptosis is to inhibit protein
translation by phosphorylation of the eIF-2
, PKR has also been found
to associate with STAT1 (9)
and to phosphorylate p53
(10)
, as well as mediate signaling through nuclear
factor-B (NF-B), likely via interaction with an IB-kinase
(11)
. An up-regulation of the FAS receptor coinciding with
apoptosis is also observed in response to dsRNA in PKR-overexpressing
cells (12)
. Interestingly, although originally identified
as the factor responsible for arresting cell-free translation in
reticulocyte lysates in response to dsRNA (13
, 14)
, it has
subsequently been demonstrated that PKR can be activated in the absence
of dsRNA, notably by polyanions and protein activators
(15)
. PKR expression is induced 5–10-fold by type I
interferons but is present at significant basal levels in their
absence. PKR activity is tightly controlled not only at the
transcriptional level but also by interaction with protein
inhibitors and activators, such as p58 and PACT, respectively
(15
, 16)
.
In this study, we provide novel evidence that human and mouse PKR are translocated to the nucleus after DNA damage, facilitate cisplatin adduct removal, and oppose apoptosis in response to genotoxic stress. Previously characterized PKR-knockout MEFs show hypersensitivity to bulky adducts and increased susceptibility to apoptosis after cisplatin treatment.
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Materials and Methods |
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TopABSTRACTIntroductionMaterials and MethodsResultsDiscussionREFERENCES |
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MEM supplemented
with 10% heat-inactivated calf serum (MDA-MB231) or in DMEM
supplemented with 10% heat-inactivated fetal bovine serum (MEFs) and
100 units/ml penicillin G and 100 units/ml streptomycin. Cells were
maintained in 5% CO2/95% air at 37°C in a
humidified incubator.
Retroviral Expression of PKR.
A bicistronic retroviral vector (17)
based on the Moloney
murine leukemia virus was used for the expression of PKR. The empty
vector or vector containing the coding sequence of wild-type PKR were
transfected by Lipofectamine into the 
2 producer cell line. Stable
producer cells were selected 1 week after daily exposure to 1 µg/ml
of puromycin as described earlier (17)
. Retroviral
infection of PKR-/- cells was performed by
filtering the producer cell culture supernatant through a 0.45 µm
filter and by adding Polybrene to a final concentration of 80 µl/10
ml 48 h after infection; cells were selected until drug-resistant
colonies were visible.
Cytotoxicity Assay.
For drug cytotoxicity assays, exponentially growing cells
(1–3 x 103
cells/well) were
seeded in 96-well plates. The next day, cells were exposed continuously
to cisplatin (Oncology Pharmacy at the Jewish General Hospital),
Adriamycin, or melphalan (Sigma Chemical Co., St. Louis, MO) for
96 h. Cell survival was evaluated by the MTT assay using MTT
(Sigma). The absorbance was measured at 570 nm with a microplate reader
(Bio-Rad). For UVC, 5000 cells/3 ml were seeded in six-well plates.
After 16 h, medium was removed, and cells were covered with 0.5 ml
of PBS and irradiated using a UV source. PBS was then removed, and
cells were collected and cultured in 96-well plates as described above.
Apoptosis Assay.
Cells were seeded at 1 x 106
cells/T75 cm2
plate and continuously exposed to
cisplatin for 72 h. Briefly, cells were collected by
trypsinization and washed twice with PBS and then diluted to
1 x 106 cells/100 µl of PBS and
placed in a 96-well plate. After fixation, cells were permeabilized
with 1% Triton X-100 in 0.1% sodium citrate and labeled in 50
µl/well TUNEL reaction mixture (Boehringer Mannheim In
Situ Cell Death Detection kit; Laval, Quebec, Canada) at 37°C in
the dark for 1 h. Cells were then washed three times with 1% BSA
in PBS and resuspended in 500 µl of PBS for analysis by flow
cytometry.
Cisplatin Accumulation, DNA Adduct Formation, and Removal.
Cells (5 x 105) were seeded in
T75 cm2
flasks. When cells reached 80–90%
confluence, they were treated with 25 µM cisplatin for
3 h in serum-free medium. Treatment was stopped by washing cells
three times with a solution of cold PBS. Cells were collected
immediately to determine the total intracellular accumulation of
cisplatin, using total cell extract or genomic DNA extract to measure
the initial amount of DNA adducts formed. Cells from duplicate flasks
were maintained in culture in drug-free complete medium to allow DNA
repair. At the indicated times, total DNA was isolated using DNAzol
solution (Life Technologies, Inc.) added directly to the flasks.
Platinum-DNA was determined by injecting a volume of 20 µl of sample
into a pyrocoated graphite cuvette using a Hitachi polarized Zeeman
Model z-8100 flameless atomic absorption spectrophotometer. A
calibration curve was established using standard platinum solutions.
Total cisplatin accumulation was expressed per µg protein, whereas
the amount of DNA adducts was expressed per µg of DNA. Comparisons
between DNA adducts in PKR-deficient and PKR-proficient cells were done
by the Student’s t test.
Immunoblotting.
Total proteins from PKR+/+,
PKR-/-, and PKR-/-
transduced with empty or wild-type PKR were collected from subconfluent
cells, using a lysis buffer [0.5% NP40, 10 mM Tris-HCl
(pH 8.0), 50 mM KCl, and 1 mM orthovanadate]
containing protease inhibitors. Proteins (50 µg) were resolved by
10% SDS-PAGE and transferred into nitrocellulose (Bio-Rad) in transfer
buffer [25 mM Tris-HCl (pH 7.5), 190 mM
glycine, and 20% v/v methanol]. Transfer of proteins was confirmed by
staining with Ponceau S (0.02% w/v). The membrane was then blocked
with 10% (w/v) skim milk in 1x PBS and then incubated with the mouse
monoclonal antibody against mouse PKR (B-10; Santa Cruz Biotechnology,
Santa Cruz, CA). Blots were washed three times with 1x PBS and
incubated with goat antimouse (Bio-Rad) immunoglobulin. Immunocomplexes
were revealed using enhanced chemiluminescence detection reagent
(Amersham).
Immunofluorescence Microscopy.
Cells were plated on coverslips 2 days before each experiment at a
concentration of 30,000–50,000 cells/35-mm dish. Cells were treated
with 10–20 µM of cisplatin for 60 min, fixed by the
addition of precooled (-80°C) methanol:acetone (80%:20% v/v)
directly to the coverslips, and then placed at -20°C for 15 min.
After fixation, the cells were rinsed extensively with PBS (pH 7.4) and
then incubated for 1 h with PBS containing 2% BSA, 2% normal
goat serum, and 0.2% gelatin at room temperature to reduce nonspecific
binding. The cells were then incubated with primary antibodies (rabbit
anti-PKR K-17, Santa Cruz Biotechnology; mouse anti--tubulin, ICN,
Mississauga, Ontario, Canada) for 1 h at room temperature. After
washing, cells were incubated with secondary antibodies (Jackson
Immunoresearch Laboratories, West Grove, PA) for 30 min at room
temperature with Texas Red goat antirabbit to reveal anti-PKR and
incubated with Cy2 goat antimouse to reveal anti--tubulin. After
labeling, the coverslips were mounted in Airvol (Air Products and
Chemicals, Inc., Allentown, PA) and viewed with a Zeiss Axiophot
fluorescent microscope equipped with 63x Plan Apochromat objectives
and selective filters. Confocal analyses were performed with a Zeiss
LSM 410 inverted confocal microscope (Institut Universitaire de
Geriatrie de Montreal, Montreal, Quebec, Canada).
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Results |
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TopABSTRACTIntroductionMaterials and MethodsResultsDiscussionREFERENCES |
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, PKR-/- cells
showed approximately 3–4-fold sensitization to cisplatin, 2–3-fold
sensitivity to melphalan, and approximately 5-fold sensitization to UV
radiation. Interestingly, we observed no difference in sensitivities of
these cell lines to Adriamycin. To determine whether the observed
differences in cytotoxicity correlate with induction of apoptosis,
we exposed both lines to 5 or 10 µM cisplatin for 48 h and then examined DNA fragmentation by a fluorescence-activated cell
sorter TUNEL assay. Fig. 2
demonstrates
significantly higher levels of cisplatin-induced apoptosis in the
PKR-/- cells.
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demonstrates that there were no significant differences in either
cisplatin uptake (147 ± 27 and 135 ± 17
pg/µg total protein for PKR-/- and
PKR+/+, respectively) or adduct formation
(29.9 ± 2.8 and 28.8 ± 3.7 pg/µg DNA
for PKR-/- and PKR+/+,
respectively) between the two cell lines. Strikingly, however, the
kinetics of adduct removal are significantly impeded in
PKR-/- cells. After a 4-h incubation in
drug-free medium, PKR+/+ cells had removed
1.5-fold more adducts than their PKR-/-counterparts (90% versus 60% of adducts removed,
respectively). By 8 h of incubation in drug-free medium, the
PKR+/+ cells had removed virtually all adducts,
whereas the PKR-/- cells had removed only 66%
of adducts (Fig. 3B)
. The specificity of these effects is
confirmed by restoration experiments in which
PKR-/- cells were stably transfected with
either wild-type PKR (PKR-/- + WT) or with
empty vector (PKR-/- + empty vector), using
a retroviral expression system. PKR is expressed to a lower level than
the endogenous protein of PKR+/+ cells (Fig. 4a)
and was also found to be
phosphorylated (data not shown). Restoration of PKR confers resistance
to cisplatin and UVC (Fig. 4, b and c)
. However,
this resistance is not restored to the same level as that observed in
PKR+/+ cells, possibly because of the lower
expression level of wild-type PKR in
PKR-/- + WT cells. Although neither of these transfections alters cisplatin uptake or adduct formation,
by 8 h PKR-/- + WT cells show 
50%
increase in repair capacity relative to
PKR-/- + empty vector (Fig. 4, d and e)
. Therefore, the amount of PKR expressed in
PKR-/- cells is capable of partially restoring
the capacity of PKR-/- + WT cells to remove
cisplatin adducts.
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B-kinase. It also interacts with the nuclear
proteins p53 and DRBP76. To gain insight into whether the observed
differences in repair capacity were the result of a PKR-mediated
signaling cascade or direct PKR action in the nucleus, we used
immunofluorescence to observe the localization of PKR in response to
cisplatin. As shown in Fig. 5
, untreated
PKR+/+ MEFs (A) and MDA-MB231 (a
human breast adenocarcinoma cell line; B) show diffuse
cytoplasmic labeling and nuclear exclusion of PKR. Upon treatment with
cisplatin, however, PKR rapidly localizes to the nucleus (Fig. 5)
. This
translocation was not observed in PKR-/-
cells.
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Discussion |
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TopABSTRACTIntroductionMaterials and MethodsResultsDiscussionREFERENCES |
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(18)
. It has also been
reported to interact with STAT1, perhaps limiting its DNA binding and
transactivation in the absence of PKR activation by IFN
(10)
. Furthermore, PKR interacts with p53, and
PKR-deficient cells show a reduction in p53 phosphorylation and
induction of p53-mediated transcription upon DNA damage. Interestingly,
this reduction in phosphorylation was seen after treatment with
Adriamycin or 
-radiation but not UV (19)
. Our study demonstrates that, relative to PKR+/+ MEFs, PKR-/- are hypersensitive to melphalan, UV, and cisplatin but not to Adriamycin. Neither differences in uptake nor adduct formation contribute to the cisplatin-sensitive phenotype of PKR-/- cells, whereas the removal of these adducts is severely impeded. To preclude the possibility that the genetic background of the wild-type and knockout cells was responsible for the observed phenotype, we restored expression of PKR in the PKR-/- cells and found that its expression does indeed confer resistance to cisplatin. The partial restoration of cisplatin resistance compared with that observed with isogenic PKR+/+ can be attributed to differences in the level of PKR expression. Although we used a highly efficient retroviral system to express PKR, the amount obtained is significantly lower than the endogenous level expressed within PKR+/+ cells. Given that no significant difference in intracellular accumulation of cisplatin was observed between PKR+/+ and PKR-/-, it is logical to speculate that decreased removal of cisplatin-adducts in PKR-/- may be attributable to impaired DNA repair or associated cell cycle checkpoint mechanisms.
Bulky adducts, such as those induced by UV and cisplatin, are substrates for NER (reviewed in Ref. 20 ). Recognition of damage results in sequential recruitment of TFIIH, XPF, and XPG. Assembly of the repair complex then leads to 3' and 5' incisions around the site of damage and excision of a 28–32-base fragment, leaving a gap that is then filled and ligated. This repair process has two classical branches: TCR and GGR. TCR is mediated by stalling of transcription at the site of bulky lesions. It is possible that such stalling facilitates secondary structure formation in the nascent mRNA, allowing binding of PKR or other double-stranded RNA binding motif proteins. GGR occurs after damage sensing and recognition, likely by a combination of XPC, XPA, and/or RPA, and the subsequent recruitment of the several other repair proteins including TFIIH (reviewed in Ref. 20 ). The adduct removal assays presented here do not, however, distinguish between GGR and TCR; it is not known whether PKR is involved in one or both branches of NER nor whether RNA is required. To date, no direct interaction has been demonstrated between PKR and any proteins known to be involved in NER, with the exception of p53. Interestingly, PKR localization to the nucleus is unimpaired in p53-null SaOS-2 (data not shown), suggesting that PKR trafficking in response to bulky adduct damage is p53-independent. It is also interesting to recall that p53 phosphorylation in response to UV is unaltered in PKR knockout MEFs (19) .
It is also worth mentioning that another protein that is induced in response to viral infection, IRF-7, has been shown recently to localize to the nucleus after genotoxic stress. The activation and localization of IRF-7 are reported to be mediated by c-Jun NH2-terminal kinase 1 (JNK1) and mitogen-activated protein kinase kinase-4 (MKK4) (21) , which is also notable because c-Jun NH2-terminal kinase inhibition reportedly sensitizes cells to cisplatin (22) . Furthermore, IRF-1, which is required for basal, but not inducible, expression of PKR (6) , may modulate p53 stability in response to DNA damage. Interestingly, IRF-1 deficiency resulted in accelerated p53 stabilization after genotoxic insult and reduced DNA repair activity after UV treatment (23) . This is consistent with a report that p53-induced apoptosis may occur via modulation of specific helicase activities (24, 25, 26) . Thus, it seems plausible that, in response to bulky adduct DNA damage, PKR may modulate NER activity via its previously reported interactions with p53 (10 , 19) , perhaps reducing the p53 interaction with the TFIIH helicases XPB and XPD, thereby promoting repair and opposing apoptosis.
PKR-deficient cells have been reported previously to have a defect in the G1-S checkpoint induced by Adriamycin treatment (19) . Our unpublished observations indicate a similar defect in the G2-M checkpoint in response to cisplatin; the role of these checkpoint defects in the observed bulky adduct-sensitive phenotype must also be examined. In summary, our results provide compelling evidence that PKR is involved in a novel pathway facilitating repair and opposing apoptosis in response to bulky adduct DNA damage.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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1 This work was supported by the Medical Research
Council of Canada (to M. A. A-J.) and in part by grants from the
Canadian Breast Cancer Research Initiative (to M. A. A-J.) and the
National Cancer Institute of Canada (to A. E. K.). 
2 These authors have contributed equally to this
study.
3 To whom requests for reprints should be
addressed, at Lady Davis Institute for Medical Research, Room 523, 3755
Chemin Cote Ste-Catherine, Montreal, Quebec, H3T 1E2 Canada. E-mail: mdaj{at}musica.mcgill.ca
4 The abbreviations used are: PKR, double-stranded
RNA-dependent protein kinase; STAT1, signal transducers and activators
of transcription 1; dsRNA, double-stranded RNA; MTT,
3-(4,5-dimethylthiazo-2-yl)-2,5-diphenyltetrazolium bromide; UVC, UV
light C; MEF, mouse embryonic fibroblast; TUNEL, terminal
deoxynucleotidyltransferase-mediated nick end labeling; NER, nucleotide
excision repair; IRF, interferon regulatory factor; TCR, transcription
coupled repair; GGR, global genome repair; TFII, transcription factor
II; XP, xeroderma pigmentosum.
Received 6/22/00. Accepted 10/25/00.
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REFERENCES |
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TopABSTRACTIntroductionMaterials and MethodsResultsDiscussionREFERENCES |
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B activation by double-stranded-RNA-activated protein kinase (PKR) is mediated through NF-B-inducing kinase and IB kinase. Mol. Cell. Biol., 20: 1278-1290, 2000. kinase from rabbit reticulocytes. J. Biol. Chem., 256: 7638-7641, 1981. subunit of eukaryotic initiation factor 2 (eIF-2) in reticulocyte lysates. Proc. Natl. Acad. Sci. USA, 77: 832-836, 1980.This article has been cited by other articles:
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  U. von Holzen, A. Pataer, U. Raju, D. Bocangel, S. A. Vorburger, Y. Liu, X. Lu, J. A. Roth, B. B. Aggarwal, G. N. Barber, et al. The Double-Stranded RNA-Activated Protein Kinase Mediates Radiation Resistance in Mouse Embryo Fibroblasts through Nuclear Factor {kappa}B and Akt Activation Clin. Cancer Res., October 15, 2007; 13(20): 6032 - 6039. [Abstract] [Full Text] [PDF]
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 A.M. Codrington, B.F. Hales, and B. Robaire Exposure of male rats to cyclophosphamide alters the chromatin structure and basic proteome in spermatozoa Hum. Reprod., May 1, 2007; 22(5): 1431 - 1442. [Abstract] [Full Text] [PDF]
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 S. Li and A. E. Koromilas Dominant Negative Function by an Alternatively Spliced Form of the Interferon-inducible Protein Kinase PKR J. Biol. Chem., April 20, 2001; 276(17): 13881 - 13890. [Abstract] [Full Text] [PDF]
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L. M. Parker, I. Fierro-Monti, and M. B. Mathews Nuclear Factor 90 Is a Substrate and Regulator of the Eukaryotic Initiation Factor 2 Kinase Double-stranded RNA-activated Protein Kinase J. Biol. Chem., August 24, 2001; 276(35): 32522 - 32530. [Abstract] [Full Text] [PDF]
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) and
PKR-/- (•) cells were exposed to
cisplatin, melphalan, or Adriamycin for 96 h or to UVC, and then
cytotoxicity was assayed by MTT. PKR-/-
cells were significantly more sensitive to bulky adduct-inducing agents
(a–c) than PKR-/- cells
but showed no increase in sensitivity to Adriamycin (which induces
double strand breaks; d). Bars, SEM.
