From the Life Sciences Division, Los Alamos National
Laboratory, Los Alamos, New Mexico 87545, the § National
Cancer Center Research Institute, Tokyo 104-0045, Japan, the
¶ National Institute of Radiological Sciences, Chiba 263-8555, Japan, and the
Memorial Sloan-Kettering Cancer Center, New
York, New York 10021
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ABSTRACT |
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Phosphorylation at serine 15 of the human p53
tumor suppressor protein is induced by DNA damage and correlates with
accumulation of p53 and its activation as a transcription factor. The
DNA-dependent protein kinase (DNA-PK) can phosphorylate
serine 15 of human p53 and the homologous serine 18 of murine p53
in vitro. Contradictory reports exist about the requirement
for DNA-PK in vivo for p53 activation and cell cycle arrest
in response to ionizing radiation. While primary SCID (severe combined
immunodeficiency) cells, that have defective DNA-PK, show normal p53
activation and cell cycle arrest, a transcriptionally inert form of p53
is induced in the SCID cell line SCGR11. In order to unambiguously
define the role of the DNA-PK catalytic subunit (DNA-PKcs) in p53
activation, we examined p53 phosphorylation in mouse embryonic
fibroblasts (MEFs) from DNA-PKcs-null mice. We found a similar pattern
of serine 18 phosphorylation and accumulation of p53 in response to
irradiation in both control and DNA-PKcs-null MEFs. The induced p53 was
capable of sequence-specific DNA binding even in the absence of
DNA-PKcs. Transactivation of the cyclin-dependent-kinase
inhibitor p21, a downstream target of p53, and the G1 cell
cycle checkpoint were also found to be normal in the DNA-PKcs When cells are exposed to DNA-damaging agents, there is
accumulation of the p53 tumor suppressor protein and its activation as
a transcription factor. This ultimately results in the arrest of cell
cycle progression until the damage is repaired. Human p53 is
phosphorylated within its transactivation domain at serine 15 in
response to DNA damage (1). Serine 15 phosphorylation results in
reduced interaction of p53 with its negative regulator, the oncoprotein
Mdm2, which, in turn, leads to the stabilization and activation of p53
(2). Subsequent up-regulation of downstream target genes by p53,
particularly the cyclin-dependent-kinase inhibitor p21,
results in the arrest of cells in the G1 phase of the cell
cycle (3).
The DNA-dependent protein kinase
(DNA-PK)1 consists of a
heterodimeric DNA-binding complex of Ku70 and Ku80 and a large
catalytic subunit, DNA-PKcs. DNA-PKcs is a member of a subgroup of the
phosphatidylinositol kinase superfamily, other members of which
function in DNA damage responses and/or cell cycle control (4).
DNA-PKcs is a serine/threonine protein kinase that is activated by DNA
double-strand breaks (DSBs). DNA-PK is required for repair of DNA DSBs,
and cells deficient in DNA-PKcs are hypersensitive to ionizing
radiation (IR) and radiomimetic drugs.
DNA-PK has been an attractive candidate for a molecule that activates
p53 in response to ionizing radiation thereby linking DNA damage to
cell cycle arrest. DNA-PK phosphorylates human p53 at serine 15 and
murine p53 at the homologous serine 18 residue in vitro (5,
6). Phosphorylation of p53 at serine 15 by purified DNA-PK leads to
reduced interaction of p53 with Mdm2 in vitro and correlates
with the stabilization of p53 and its activation as a transcription
factor (2). It has recently been reported that a SCID (severe combined
immunodeficiency) cell line SCGR11 accumulates a transcriptionally
inactive form of p53 upon irradiation (7). As SCID cells have defective
DNA-PKcs (8), it would appear that DNA-PK indeed acts upstream of p53
in response to DNA damage.
However, the role of DNA-PK in p53 activation has been called into
question by reports of normal p53 induction and cell cycle arrest in
response to IR in primary cells from SCID mice (9-12). Moreover, the
ATM protein, encoded by the gene responsible for the human genetic
disorder ataxia telangiectasia (AT), is required for the
phosphorylation of p53 at serine 15 in vivo in response to
IR (13, 14). AT cells have normal levels of DNA-PK (15), yet show
reduced and delayed p53 serine-15 phosphorylation in response to IR
(1). These results, on the other hand, suggest that while DNA-PK may
phosphorylate p53 in vitro, it may not play an essential
role in the activation of p53 nor can it substitute for the ATM protein
in response to IR.
In order to definitively delineate the role of DNA-PK in p53 activation
we utilized cells derived from DNA-PKcs-null mice. These cells, unlike
primary SCID cells (7), are completely deficient in DNA-PK kinase
activity (16). Transformed cell lines like SCGR11 often have mutations
in p53. Therefore, studies were performed on early passage fibroblasts
derived from 13.5-day-old mouse embryos. Control and DNA-PKcs-null
cells were irradiated and compared with respect to accumulation of p53
protein, serine 18 phosphorylation of p53, sequence-specific DNA
binding by p53, up-regulation of p21 gene expression, and integrity of
the G1 cell cycle checkpoint.
Establishment of DNA-PKcs +/ Reverse Transcription-PCR (RT-PCR) and Western Blotting--
For
RT-PCR, total RNA was prepared from mock-irradiated or irradiated MEFs
at 2 h post-irradiation using the Qiagen RNeasy kit (Qiagen,
Chatsworth, CA). After digestion of contaminating genomic DNA by DNase
I (Ambion, Austin, TX), cDNA synthesis was carried out with the
Superscript preamplification system (Life Technologies, Inc.) according
to the included protocol. PCR primers used for RT-PCR were MD-3
(5'-ATCAGAAGGTCTAAGGCTGGAAT-3') and MD-5 (5'-CGTACGGTGTTGGCTACTGC-3')
for amplification between exons 1 and 4 of DNA-PKcs and GA-5
(5'-AGAAGACTGTGGATGGCCCC-3') and GA-3 (5'-AGGTCCACCACCCTGTTGC-3')
for control glyceraldehyde 3-phosphate dehydrogenase (GAPDH) amplification.
Whole-cell extracts for DNA-PKcs Western blotting were prepared from
mock-irradiated and irradiated MEFs at 2 h post-irradiation as
described previously (17). The protein concentration of extracts was
determined by Bradford analysis using bovine serum albumin as a
standard. Western blot analysis of DNA-PKcs was performed as described
previously (18) using anti-DNA-PKcs monoclonal antibodies (42-26)
(NeoMarkers, Fremont, CA).
SDS extracts for p53, p53 phosphoserine 15, and actin Western blotting
were prepared from mock-irradiated or irradiated cells as described
previously (19). The antibodies used for Western blotting are anti-p53
PAb122 (PharMingen, San Diego, CA), anti-phosphoserine 15 p53 peptide
(2), and anti-actin (C-11) (Santa Cruz Biotechnology, Inc., Santa Cruz, CA).
Electrophoretic Mobility Shift Assays (EMSAs)--
Cell extracts
were prepared from mock-irradiated and irradiated MEFs at 2 h
post-irradiation as described previously (17). DNA binding was analyzed
by EMSA using the 32P-radiolabeled p53 consensus sequence
5'-AGCTTAGACATGCCTAGACATGCCAAGCT-3'. A typical binding reaction
contained 1.5 µg of cell lysate, 0.25 µl of 0.1 M
dithiothreitol, 1 µl of 32P-radiolabeled DNA (1 ng/µl),
1.2 µl of glycerol, 0.5 µl of PAb421 (Calbiochem), and
Tris-buffered saline (25 mM Tris, pH 7.5, 130 mM NaCl, 3 mM KCl) to 10 µl final volume as
described previously (7, 20). Reactions were incubated for 30-60 min
at 23 °C and electrophoresed in a nondenaturing Tris borate-EDTA
polyacrylamide gel.
Quantitative RT-PCR--
For quantitative RT-PCR, equal amounts
of total RNA prepared from mock-irradiated or irradiated MEFs at 4 h post-irradiation were used for cDNA synthesis as described
previously in this section. The cDNAs were then amplified for p21
through 15 PCR cycles using the primers 5'-CGGTCCCGTGGACAGTGAGCAG-3'
and 5'-GTCAGGCTGGTCTGCCTCCG-3'. The GAPDH primers GA-5 and GA-3,
described previously in this section, were used as a normalizing
control. PCR products were electrophoresed through a 2% Tris
acetate-EDTA-agarose gel, transferred to Hybond-N+ nylon
membranes (Amersham Pharmacia Biotech) and were hybridized with
32P-radiolabeled probes specific to the amplified p21 or
GAPDH fragments: 5'-CCCGAGAACGGTGGAACTTTGACTTCGTCA-3' for p21 and
5'-AGTATGATGACATCAAGAAGGTGGTGAAGC-3' for GAPDH. After hybridization,
the blots were washed in buffer containing 0.5 × SSC and 0.1%
SDS at 42 °C and autoradiographed. The autoradiographs were scanned
and the bands quantified using NIH Image.
BrdUrd Labeling and Flow Cytometric Analyses--
Asynchronously
growing control and irradiated MEFs were continuously labeled with 10 µM BrdUrd after 6 Gy of Establishment and Characterization of Embryonic Fibroblasts from
DNA-PKcs-null Mice--
We have generated DNA-PKcs-null mice by
disrupting the DNA-PKcs gene by homologous recombination (16). MEFs
were isolated from DNA-PKcs +/ Accumulation and Phosphorylation of p53 in Response to
IR--
DNA-PKcs +/ Sequence-specific DNA Binding by p53 from DNA-PKcs Up-regulation of p21 Gene Expression upon Irradiation of Both
DNA-PKcs +/ The G1 Cell Cycle Checkpoint Is Intact in the DNA-PKcs
In this paper we present evidence proving that DNA-PKcs is not
essential for p53 activation and cell cycle arrest in response to IR.
Upon irradiating DNA-PKcs-null MEFs, we observe normal p53 accumulation
and serine 18 phosphorylation, sequence-specific DNA binding by p53,
transactivation of p21 gene expression, and normal G1 cell
cycle arrest in the absence of DNA-PKcs.
Earlier reports on p53 activation in the absence of DNA-PKcs were based
upon experiments with primary cells from SCID mice (9-12). However,
primary SCID cells have been reported to retain residual DNA-PK kinase
activity (7). This could be because the SCID mutation resides just
downstream of the conserved kinase motifs of the DNA-PKcs gene
resulting in a truncated product missing only 83 amino acid residues
from the extreme carboxyl-terminal end (8). In order to avoid the
ambiguity associated with SCID cells, we established MEFs derived from
DNA-PKcs-null mice as these cells are completely deficient in DNA-PKcs
and, unlike many transformed cell lines, would not have mutations in p53.
The DNA-PKcs-null mice, generated by disrupting the DNA-PKcs gene by
homologous recombination, show severe immunodeficiency and radiation
hypersensitivity (16). Cells from the DNA-PKcs-null mice have no
detectable DNA-PKcs protein or kinase activity (16). The DNA-PKcs +/ Phosphorylation of human p53 within its transactivation domain at
serine 15 or murine p53 at serine 18 is an early and important step in
the activation and accumulation of this protein in response to IR (24).
This phosphorylation event could contribute to both increased p53
half-life and increased transcriptional activity by decreasing the
ability of the negative regulator Mdm2 to bind to p53 (2). However,
none of the earlier reports describing the involvement or
noninvolvement of DNA-PK in p53 activation have focused on this
phosphorylation event. Our results demonstrate that DNA-PKcs, unlike
ATM, is not essential for the phosphorylation or accumulation of p53 in
response to IR.
It is interesting that phosphorylation and accumulation of p53 are
actually enhanced in the DNA-PKcs Our observations regarding induction of sequence-specific DNA binding
by p53 upon irradiation of the DNA-PKcs Woo et. al. (7) also reported that cytoplasmic extracts from
p53 activation in response to IR leads to transactivation of the
cyclin-dependent-kinase inhibitor p21 and G1
cell cycle arrest (24). In order to correlate the observed p53
accumulation, phosphorylation, and DNA binding with its activation as a
transcription factor, we looked at the induction of p21 gene expression
upon irradiation and the integrity of the G1 cell cycle
checkpoint in the DNA-PKcs Our observations involving DNA-PKcs /
MEFs. Our results demonstrate that DNA-PKcs, unlike the related ATM
protein, is not essential for the activation of p53 and G1
cell cycle arrest in response to ionizing radiation.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
and
/
Mouse Embryonic
Fibroblasts (MEFs) and Irradiation of Cells--
Primary fibroblasts
were isolated from 13.5-day-old mouse embryos. The embryos were
genotyped by PCR which distinguishes the endogenous from the targeted
DNA-PKcs allele (16). Cells were maintained in a humidified atmosphere
with 5% CO2 in
-minimal essential medium supplemented
with 20% fetal calf serum, 100 units/ml penicillin, and 100 µg/ml
streptomycin. Cells were grown to 80% confluence and irradiated with
137Cs
-rays at the rate of 4.2 Gy/min to achieve a
cumulative dose of 8 Gy for all experiments with the following
exception. For flow cytometry, 50% confluent cells were irradiated
with a cumulative dose of 6 Gy.
-irradiation. Cells were then
harvested at 6 h post-irradiation and fixed in 80% ethanol. Fixed
cells were simultaneously stained with Hoechst 33342 (HO) and
mithramycin (MI) and analyzed by multiparameter flow cytometry (21).
Cellular DNA content is proportional to the MI fluorescence and BrdUrd
incorporation to the difference between the MI and HO fluorescence
signal per cell (MI-HO). Bivariate histograms of MI-HO
versus MI fluorescence were obtained for 2 × 104 events as described (21). Bivariate contour histograms
in the figures were drawn with WinMDI v2.4 (Joseph Trotter, The Scripps Research Institute, La Jolla, CA).
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
and
/
sibling embryos. The
genotype of the embryos was determined by PCR (16) that distinguishes
endogenous from the disrupted DNA-PKcs allele (data not shown). The
presence of intact DNA-PKcs transcripts in the +/
MEFs and its
absence in the
/
MEFs was confirmed by reverse transcription-PCR
(RT-PCR). RT-PCR products between exon 1 and exon 4 of DNA-PKcs were
clearly absent in the
/
MEFs (Fig.
1A). Western blotting revealed
the presence of equal amounts of DNA-PKcs protein in control and
irradiated +/
MEFs, but no detectable protein in the
/
MEFs (Fig.
1B). The +/
and
/
MEFs were also characterized by low
dose rate irradiation with 137Cs
-rays (1.4 Gy/day for
10 days). After irradiation, the cells were allowed to recover for 10 days. While we observed growing DNA-PKcs +/
MEFs, no surviving
DNA-PKcs
/
MEFs were observed after 10 days.
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Fig. 1.
Complete absence of DNA-PKcs expression in
the DNA-PKcs /
MEFs. A, RT-PCR of region between
exons 1 and 4 of DNA-PKcs RNA from DNA-PKcs +/
and
/
MEFs. Total
RNA was isolated from mock-irradiated (C) or irradiated
(IR) MEFs. PCR reactions were performed with (+) or without
(
) RT. RT-PCR for GAPDH was performed as a normalizing control.
B, Western blot analysis of DNA-PKcs +/
and
/
MEFs.
Whole cell extracts were prepared from mock-irradiated (C)
or irradiated (IR) MEFs. Anti-DNA-PKcs monoclonal antibody
was used for detection.
and
/
MEFs were grown to 80% confluence,
irradiated, and harvested at 0, 2, 4, and 8 h post-irradiation.
SDS extracts prepared from the irradiated cells were examined for p53
accumulation and phosphorylation by Western blotting using anti-p53
antibody and anti-phosphoserine 15 p53 peptide antibody. The
anti-phosphoserine 15 antibody can recognize phosphorylated serine 15 of human p53, as well as the phosphorylated form of the homologous
serine 18 of murine p53, but not unphosphorylated p53 (2). We observed
normal accumulation of p53 in response to IR in both DNA-PKcs +/
and
/
MEFs (Fig. 2, top
panel). Serine 18 phosphorylation was more transient, lasting
between 2 and 4 h, with maximum levels of phosphorylation at
2 h post-irradiation (Fig. 2, middle panel). Ser-18
phosphorylation of p53 was not impaired in the DNA-PKcs
/
MEFs.
Moreover, both accumulation and phosphorylation of p53 were enhanced in
the absence of DNA-PKcs.
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Fig. 2.
Phosphorylation and accumulation of p53 in
DNA-PKcs +/ and
/
MEFs. SDS extracts were prepared from
DNA-PKcs +/
and
/
MEFs at 0, 2, 4, and 8 h after
irradiation. Western blot analysis was carried out using anti-P53
antibody (top panel), anti-phosphoserine 15 p53 peptide
antibody (middle panel), and anti-actin antibody
(bottom panel) as normalizing control.
/
MEFs--
Cell extracts from mock-irradiated and irradiated DNA-PKcs
+/
and
/
MEFs were assayed for p53 sequence-specific DNA binding by EMSA using double-stranded oligonucleotides bearing the consensus p53 DNA-binding motif (Fig. 3). Binding
reactions were carried out in the presence of a specific anti-p53
antibody PAb421 that activates sequence-specific DNA binding by p53
(22). The bands indicated by an arrow in Fig. 3 represent
sequence-specific DNA binding by p53 as they are not obtained in the
absence of PAb421 (Fig. 3, lane 6) or in the presence of an
unrelated antibody (Fig. 3, lane 7). Enhancement of
sequence-specific DNA binding by p53 was observed upon irradiation of
the +/
MEFs (Fig. 3, compare lanes 2 and 3) and also upon
irradiation of the
/
MEFs (Fig. 3, compare lanes
4 and 5).
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Fig. 3.
Sequence-specific DNA binding by p53 from
DNA-PKcs /
MEFs. Cell extracts from mock-irradiated
(C) or irradiated (IR) DNA-PKcs +/
and
/
MEFs were assayed for p53 DNA binding by EMSA. Cell extract was not
added to the binding reaction in lane 1. The p53-specific
antibody PAb421 was added to the binding reactions in lanes
2-5. The last two lanes are controls and are identical to
lane 5 except that no antibody was added in lane
6, and anti-actin antibody was added instead of PAb421 in
lane 7. Bands indicated by the arrow represent
sequence-specific DNA binding by p53 as they are absent in the control
lanes.
and
/
MEFs--
Activated p53 can transactivate the
cyclin-dependent-kinase inhibitor p21 (23). Therefore, in
order to test whether the observed phosphorylation, accumulation and
DNA binding of p53 corresponded to activation of transcription in
vivo, we quantitated the induction of p21 messenger RNA upon
irradiation of DNA-PKcs +/
and
/
MEFs by quantitative RT-PCR. We
found that the +/
and
/
MEFs are indistinguishable with respect
to the enhancement (4.5-5-fold) of p21 gene expression upon
irradiation (Fig. 4, A and
B).
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Fig. 4.
DNA-PKcs +/ and
/
MEFs are
indistinguishable with regard to induction of p21 gene expression upon
irradiation. A, quantitative RT-PCR to determine
relative p21 expression levels in mock-irradiated (C) and
irradiated (IR) DNA-PKcs +/
and
/
MEFs. Total RNA was
isolated from these cells and used for cDNA synthesis using reverse
transcriptase. The cDNA was PCR amplified using p21-specific
primers, transferred to a nylon membrane, hybridized with a
p21-specific probe, and autoradiographed. Constitutively expressed
GAPDH was similarly amplified as a normalizing control. B,
relative induction of p21 upon irradiation of DNA-PKcs +/
and
/
MEFs. The autoradiograph in Fig. 1A was scanned, and the
relative p21 expression levels were plotted on the y
axis.
/
MEFs--
To investigate the effect, if any, of the absence of
DNA-PKcs on the G1 cell cycle checkpoint, DNA-PKcs +/
and
/
MEFs were mock-irradiated or irradiated at 6.0 Gy and labeled
with BrdUrd immediately after irradiation. BrdUrd incorporations at
6 h post-irradiation were quantified from multiparameter flow
cytometry measurements (21). The bivariate contour histograms (Fig.
5A) depict the distributions
of cells with different BrdUrd (MI-HO fluorescence) and DNA (MI
fluorescence) contents in the control and irradiated cultures. By
6 h post-irradiation, cells were depleted from the S phase
(boxed in Fig. 5A) in both DNA-PKcs +/
and
/
cultures indicating induction of arrest at a G1
checkpoint. The ratios of percentage of total cells in G1
to percentage of total cells in S phase (percent cells in
G1/percent cells in S) for control and irradiated cultures
were plotted (Fig. 5B). The extent of G1 arrest
was comparable for the DNA-PKcs +/
and
/
cultures (Fig.
5B).
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Fig. 5.
The G1 cell cycle checkpoint is
intact in DNA-PKcs /
MEFs. A, bivariate contour
MI-HO versus MI histograms of cells from control and 6 h post-irradiated DNA-PKcs +/
and
/
MEFs depicting the
distributions of cells with different BrdUrd and DNA contents. The MI
channel number is proportional to DNA content, and MI-HO is linearly
related to BrdUrd incorporation for signals above background. The
box depicts the position of cells in the S phase of the cell
cycle. Please note the depletion of both DNA-PKcs +/
and
/
MEFs
from the S phase compartment after irradiation. B, extent of
G1 arrest in DNA-PKcs +/
and
/
cultures. The ratios
of percentage of total cells in G1 to percentage of total
cells in S phase (percent cells in G1/percent cells in S)
for control and irradiated cultures were plotted on the y
axis.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
and
/
MEFs used for this study were extensively characterized with
respect to DNA-PKcs status and phenotype by (i) PCR (for genotyping),
(ii) RT-PCR (for confirming the presence of intact DNA-PKcs transcripts
in the +/
but not in the
/
MEFs), (iii) Western blotting (for
confirming the presence of DNA-PKcs protein in +/
MEFs and its
absence in
/
MEFs), and (iv) low dose rate irradiation (only the
/
MEFs were susceptible).
/
MEFs relative to the +/
MEFs.
Our observations are consistent with those of Araki et
al.2 who observed a
similar enhancement in transformed mouse cell lines with defective
DNA-PKcs and with those of Gurley and Kemp (11) who observed a
prolonged induction of p53 protein in the intestinal crypt cells of
irradiated SCID mice. The ATM protein can bind to DNA ends like
DNA-PKcs, and the p53 serine 15 kinase activity of ATM is stimulated by
the presence of damaged DNA.3
We speculate that in the absence of DNA-PKcs, ATM may have greater access to DNA DSBs as it no longer has to compete with DNA-PKcs for end
binding. This, coupled with the persistence of DNA DSBs in the absence
of DNA-PK, could result in enhanced activation of ATM, which may
explain the enhanced phosphorylation and accumulation of p53 observed
in the DNA-PKcs
/
MEFs.
/
MEFs are not in agreement
with those of Woo et al. (7) who fail to detect any DNA
binding using extracts from SCGR11 cells. This difference is possibly
due to the fact that p53 from SCGR11 is mutated in its DNA-binding
domain.2 The mutation is a "T" to "C" transversion
resulting in a substitution of leucine at position 191 to arginine
(DDBJ accession number AB021961). The vast majority of p53 missense
mutations are clustered within the DNA-binding region and the inability
of p53 from SCGR11 to bind to DNA would be due to the defect in its
DNA-binding domain and not, as proposed by Woo et. al. (7),
due to the absence of DNA-PKcs in these cells.
-irradiated M059J cells (that lack DNA-PKcs) could not activate in vitro-translated p53 for DNA binding (7). However, M059J cells also have drastically reduced levels of the ATM protein (25). The
ATM protein is essential for p53 activation upon irradiation and the
lack of p53 activation by MO59J cytoplasmic extracts may not be due to
the absence of DNA-PKcs but due to the reduced levels of ATM.
/
MEFs. We found that the DNA-PKcs +/
and
/
MEFs are indistinguishable with regard to fold induction of
p21 gene transcription upon irradiation. We also found that both DNA-PK
+/
and
/
cultures arrested in the G1 phase of the
cell cycle by 6 h post-irradiation. These results are consistent
with earlier reports of normal p21 gene induction and cell cycle arrest
in primary SCID cells (9-12).
/
MEFs indicate unequivocally
that DNA-PKcs, unlike the related ATM protein, is not essential for the
activation of p53 and G1 cell cycle arrest in response to
ionizing radiation. An alternate interpretation could be that DNA-PKcs
is still involved in p53 activation, as suggested by in
vitro experiments (2, 5, 6), but the loss of DNA-PKcs activity is
concealed by the presence of other p53-dependent pathways like the one involving ATM. In this regard, it should be noted that AT
cells have mutated ATM protein but contain wild type DNA-PKcs. So, if
DNA-PKcs were to play a major role in p53 activation, then p53
phosphorylation in A-T cells would not be significantly reduced and
delayed (1). Therefore, while DNA-PKcs is involved in the repair of DNA
DSBs (4, 17), our experiments clearly rule out an essential role for
DNA-PKcs in the activation of a p53-mediated DNA damage signaling pathway.
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ACKNOWLEDGEMENTS |
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We thank Melinda Henrie and Paige Pardington for their excellent technical help and Steve Yannone and Robert Cary for critically reading the manuscript.
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FOOTNOTES |
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* This work was supported in part by National Institutes of Health Grants CA-50519 (to D. J. C.) and CA-78497 and CA-56909 (both to G. C. L.) and by the Department of Energy Office of Health and Environmental Research (to D. J. C.).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: Life Sciences Division, MS 74, Lawrence Berkeley National Laboratory, 1 Cyclotron Rd., Berkeley, CA 94720. Fax: 510-486-5735.
Current address: Life Sciences Division, Lawrence Berkeley
National Laboratory, Berkeley, CA 94720.
2 R. Araki, R. Fukumura, A. Fujimori, Y. Taya, Y. Shiloh, A. Kurimasa, S. Burma, G. C. Li, D. J. Chen, K. Sato, Y. Hoki, K. Tatsumi, and M. Abe, submitted for publication.
3 G. C. M. Smith, R. B. Cary, N. D. Lakin, B. C. Hann, S.-H. Teo, D. J. Chen, and S. P. Jackson, submitted for publication.
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ABBREVIATIONS |
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The abbreviations used are: DNA-PK, DNA-dependent protein kinase; DNA-PKcs, DNA-PK catalytic subunit; DSB, double-strand break; IR, ionizing radiation; SCID, severe combined immunodeficiency; AT, ataxia telangiectasia; MEF, mouse embryonic fibroblast; Gy, gray; RT-PCR, reverse transcription-polymerase chain reaction; GAPDH, glyceraldehyde 3-phosphate dehydrogenase; EMSA, electrophoretic mobility shift assay; BrdUrd, bromodeoxyuridine; HO, Hoechst 33342; MI, mithramycin.
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