 |
INTRODUCTION |
The tumor suppressor protein, p53, plays a critical role in the
cellular response to genotoxic stress. Cells sustaining DNA damage,
such as that caused by ionizing radiation
(IR),1 develop an increase in
p53 protein levels via a post-transcriptional mechanism (1).
Co-incident with the increase in the levels of p53 is the activation of
p53-dependent transcription for a variety of genes,
including p21WAF1/cip1, GADD45 and MDM2, resulting in either
G1/S cell cycle arrest or apoptosis (2-5).
The mechanism by which the signal of DNA damage is transduced to p53
remains to be defined. Initially, it had been presumed that the effect
of the signal from DNA damage was to increase the amount of p53 protein
in the cell, which could then signal other genes in the damage response
pathway (6). More recently, it has been reported that
p53-dependent transactivation can occur in the absence of
any increase in the level of p53 (7). In addition, Haapajarvi et
al. (8) demonstrated that the effect of ultraviolet light exposure
on cells synchronized in G1 was to increase transcriptional
activity of p53 and subsequently G1 arrest, well before any
demonstrated increase in the levels of p53 protein. Furthermore, the
accumulation of p53 only occurred when the cells had progressed into
S-phase of the cell cycle. Thus, it seems likely that the signal from
DNA damage can affect both the levels of p53 and the active state of
p53 (post-translational modification). These two responses from DNA
damage to p53 could share a common signaling pathway or utilize
different pathways, as there has been no genetic basis to separate them
to date. Most recently, the use of protein kinase C inhibitors resulted
in increased levels of p53 (secondary to an increase in its half-life)
and consequent translocation to the nucleus. However, DNA damage was still needed to increase the activity of p53, as measured by a
-galactosidase transactivation reporter assay (9).
The timing of the response to DNA damage does not provide a definitive
answer to discriminate between post-translational modification and
elevated levels of p53. The levels of p53 are seen to rise by 1-2 h
after exposure to IR and reach a peak between 2 and 6 h, depending
on the cell type tested. The levels of MDM2 are found to increase
within 2-4 h of IR (10) and p21 shows a similar time course (3-11).
The fact that downstream genes are transcriptionally activated and the
resultant protein detected by 2 h suggests that the rapid response
to p53 is mediated by a post-translational modification of p53 or
modification of an inhibitory protein such as MDM2, which occurs more
rapidly than a rise in the level of p53.
The DNA-activated (or dependent) protein kinase (DNA-PK) is a nuclear
serine/threonine protein kinase that is activated in vitro
by DNA fragments (12, 13). Several observations have suggested that a
large polypeptide (initially reported to be 350 kDa, but now thought to
be approximately 460 kDa (14)) is the catalytic subunit of DNA-PK,
while a cellular DNA end-binding protein, the Ku autoantigen
(consisting of 70- and 80-86-kDa proteins), serves as the regulatory
subunit (13, 15, 16). DNA-PK has been shown to phosphorylate several
nuclear DNA-binding proteins in vitro, including the
transactivation domain of p53 (17). Recently, these observations were
expanded to show the importance of serines 15 and 37 in mediating the
response to DNA damage in vivo, and showing these sites were
phosphorylated in vitro by DNA-PK (18). Furthermore, serine
15 of p53 has been shown to be one site which is phosphorylated in
response to DNA damage (19). Mutation of the serine 15 phosphorylation
site of human p53 to alanine demonstrated impaired transactivation of
p53 and, as a consequence, impaired cell-cycle arrest at the
G1/S transition (20). However, Shieh et al. (18)
made reference to unpublished data showing constitutive and induced
phosphorylation of p53 at serine 15 in murine scid cells,
implying that other pathways distinct from DNA-PK can phosphorylate p53
at serine 15 in response to DNA damage. The ATM kinase is also a
candidate kinase for phosphorylation of serine 15, but ATM-deficient
cells also show induced phosphorylation at this site implying parallel
pathways to signal stress, such as DNA damage, via phosphorylation of
serine 15 of p53. More recent studies have shown that adding back the
ATM gene in these deficient cells results in increased phosphorylation
at the serine 15 site, using a specific phosphoserine antibody
(21).
The mouse homologue of the human gene, encoding the catalytic subunit
of DNA-PK, has been found to be mutated in the scid (severe
combined immunodeficiency) gene locus (22, 23). The mutation has been
characterized as a short deletion in the C-terminal domain of the
protein, which destabilizes the protein and results in a low level of
measurable DNA-PK activity (24). The mouse scid is a
recessive, autosomal mutation that results in the inability to produce
mature, functioning lymphocytes because of failure in V(D)J
recombination (25, 26). scid mice and scid cells in culture have been shown to be highly sensitive to IR and other agents that induce DNA double-strand breaks, suggesting that the scid gene product plays a role in DNA repair (27-30). The
other two gene products, Ku80 and Ku70, which together with the
scid gene product (DNA-PKcs) constitute the DNA-PK
holoenzyme, have also been shown to be involved in the same response
pathways, since cells containing mutant or absent Ku 80 or Ku 70 are
sensitive to IR and deficient V(D)J recombination (31-33).
A number of recent reports have shown that the p53 response and the
G1/S cell cycle checkpoint in murine scid cells
is normal (34-38), casting serious doubt about any connection between
p53 and DNA-PK. However, none of these publications looked in detail at
the kinetics of cell cycle arrest or the timing of transactivation of
p53-dependent downstream genes by observing multiple time
points following exposure to ionizing radiation or other DNA damaging agent. Furthermore, DNA damage induced phosphorylation of serine 15 of
p53 results in alleviation of the inhibition from MDM2 (18). More
recently, it has been shown that DNA-PKcs is necessary for activation
of DNA binding by p53 (in response to IR) in a gel shift assay (39).
Failure to phosphorylate p53 could produce the phenotype of
scid cells: an attenuated and perhaps delayed response
produced in proportion to the elevation of levels of p53, without the
rapid response brought about by loss of inhibition by MDM2. The effect
of DNA-PK could be directly on p53 or indirectly on an inhibitory
protein such as MDM2. It has recently been suggested that DNA-PK might
phosphorylate MDM2, which then prevents inhibition of p53 (40). These
observations are not mutually exclusive, and therefore the effect of
loss of DNA-PK activity could be a combined effect of lack of
phosphorylation of p53 (resulting in impaired transactivation) and lack
of phosphorylation of MDM2 which then continues to attenuate p53
(rather than phosphorylation which results in less inhibition of p53).
Thus, the exact role of DNA-PK in vivo remains to be
defined, as it appears to function both in DNA repair and in sensing DNA damage (41). The gene mutated in patients with ataxia
telangiectasia (ATM) produces a protein which is related to DNA-PKcs.
Cells containing mutant or absent ATM have previously been described to
have an abnormal response of p53 to IR (42). Although the precise
nature of the p53 response in ataxia telangiectasia cells has been
debated (42, 43) there now appears to be a consensus that the response is delayed and attenuated (44, 45). Therefore, we investigated the DNA
damage response pathways in mouse scid cells in order to
ascertain whether DNA-PK can regulate the function of p53 in vivo.
 |
MATERIALS AND METHODS |
Cell Culture, DNA Transfer, and Growth Selection--
p53
function was analyzed in two murine cell cultures: primary murine
embryonic (day 20-22) fibroblasts (MEF) from a normal mouse (FC) and
from a Balb-C derived scid mouse (FS). The primary cultures
were established directly from the mouse embryos obtained from the
Edwin L Steele Laboratory, Massachusetts General Hospital, as described
previously (46). These cells were cultured in Dulbecco's modified
Eagle's medium at 37 °C in humidified air with 5% CO2. The medium was supplemented with 1 mg/ml glucose, 10% fetal bovine serum, 100 units/ml penicillin, 0.1 mg/ml streptomycin, and 20 mmol of
Hepes solution (Sigma). The cells were maintained in exponential growth
in monolayer cultures, and used within 10-12 passages from their
initial establishment in cell culture. If cells were maintained in cell
culture for more than 20 passages, a significant incidence of
developing abnormal p53 function was observed (47). Retroviral constructs containing the E6 protein of human papilloma virus type
16/18 (pXIPneoE6; D. Galloway, Fred Hutchinson Cancer Center) were
transferred into FC and FS cells, and cells containing a stable
integration of the vector were selected by growth in G418 (0.5 mg/ml;
Life Technologies, Inc.).
Cell Irradiation--
Murine embryonic fibroblasts from normal
and scid mice were plated in 150-mm dishes and grown to
50-70% confluence at the time of irradiation. The tissue culture
medium was renewed 1 day prior to irradiation to remove non-viable
unattached cells. Ionizing radiation was delivered using either a
60Co source (Theratron) or a linear accelerator delivering
4MV x-rays (Varian).
Flow Cytometry and Cell Cycle Analysis--
The cell cycle at
the G1/S transition in response to IR was measured using
flow cytometry as described previously (48). In brief, exponentially
growing cells (1-5 × 105) were irradiated with 8 Gy
IR and collected at specific time points (0, 3, 6, 9, 12, and 24 h) following irradiation. The cells were harvested from the culture
dishes using trypsin/EDTA, washed twice in phosphate-buffered saline
(PBS) before suspension in 0.75 ml of ice-cold 70% ethanol, and
incubated on ice for >1 h. The cells were pelleted, washed a further
two times in PBS, and then exposed to propidium iodide (100 µg/ml)
and RNase A (500 µg/ml). DNA analysis was performed using a FACScan
flow cytometer (Becton Dickinson, San Jose, CA) emitting a 488-nm beam
(26). The G1/S transition was evaluated by the decrease in
the proportion of S-phase cells coupled with an accumulation of cells
in G1, with time after irradiation.
Cell Lysates--
Cells were harvested and lysed at 0, 3, 6, 9, 12, and 24 h after 8 Gy irradiation, in parallel with the flow
cytometric analysis. For each time point, cells were washed twice in
ice-cold PBS, then 1 ml of ice-cold lysis buffer (1% Triton X-100, 10 mM Tris, pH 7.4, 150 mM NaCl, 5 mM
EDTA, 10% glycerol, 1 mM sodium orthovanadate, 1 mM phenylmethylsulfonyl fluoride, 1 mM
A-protinin) was added and cells were scraped from the plates. The cell
suspension was incubated on ice for 30 min and then centrifuged for 10 min at 13,000 × g at 4 °C. Supernatant was then
removed and protein levels quantified using the Bio-Rad protein
quantification solution. Whole cell lysates were stored at
70 °C.
Preparation of nuclear lysates, at the same time points as the
collection of whole cell lysates, used the method described by Price
and Calderwood (49). In brief, cells were first washed twice in
ice-cold PBS, then 0.8 ml of nuclear lysis buffer (20 mM
Hepes, pH 7.8, 20% glycerol, 1.5 mM MgCl2, 0.2 mM EDTA, 10 mM NaCl, 1 mM
dithiothreitol, 1 mM phenylmethylsulfonyl fluoride, 0.1%
Triton X-100) was added and cells were scraped from the plates. The
cell suspension was incubated on ice for 5 min and then spun down at
2,000 × g for 10 min at 4 °C. Supernatant was then
removed and pellet was re-suspended in 400 µl of nuclear extraction
buffer (20 mM Hepes, pH 7.8, 20% glycerol, 1.5 mM MgCl2, 0.2 mM EDTA, 500 mM NaCl, 1 mM dithiothreitol, 1 mM
phenylmethylsulfonyl fluoride, 0.1% Triton X-100). After incubation on
ice for 30 min, the mixture was then spun at 25,000 × g for 15 min. The supernatant (containing nuclear proteins)
was then removed and the protein quantified. The lysates were stored at
70 °C.
Western Analysis--
After protein quantification, Western
analysis (immunoblot) was carried out using standard procedures with
the use of antibodies against p53 (pAb 421-Oncogene Science), p21
(Santa Cruz), and mouse MDM2 (2A10, A. J. Levine, Princeton (50)).
In summary, 50 µg of protein were loaded into each well of a 12%
SDS-polyacrylamide electrophoresis gel and electrophoresis carried out
with 1 × Tris glycine running buffer (Bio-Rad). After membrane
transfer, blocking was performed with 10% non-fat milk, 0.1%
Tween-PBS solution followed by incubation with the relevant antibodies.
Antibody detection employed the enhanced chemiluminescence (ECL,
Amersham) technique. Relative signal intensity of the protein bands was
measured using an Epson 636 scanner with transparency adapter.
Immunofluorescence--
Cells were grown on slides for 24 h
before irradiation and stained 6 h later. Cells were washed 2 times in PBS, fixed in 70% methanol, and 30% acetone for 2 min and
then washed again with PBS. The primary antibody used for p53 analysis
was pAb421 at a concentration of 10 µg/ml in PBS with 3% bovine
serum albumin. Cells were incubated at room temperature for 6 h
and then washed 3 times in PBS. The secondary antibody used was the
fluorescein-conjugated goat anti-mouse antibody (Oncogene Science). The
cells were incubated for 6 h in the dark at room temperature with
a 1:10 dilution of the secondary antibody in PBS with 3% bovine serum
albumin. Cells were then washed 3 times in PBS. Slides were prepared
using the Dako mounting solution (Oncogene), coverslipped, and stored
in the dark at 4 °C. Immunofluorescence studies always included a negative control (secondary antibody alone) as well as a positive control (U2OS cells, a cell line derived from human osteosarcoma, with
high levels of nuclear and cytoplasmic p53).
 |
RESULTS |
Cell Cycle Arrest following DNA Damage Is Delayed in Scid
Fibroblasts--
Normal fibroblasts with wild-type p53 function arrest
in the G1/S phase of the cell cycle when exposed to IR (42,
51, 52). This response is attributable to the activation of p53 by
radiation-induced DNA damage or similar lesions. If DNA-PK participates
directly in DNA damage recognition and transmits this signal to p53,
the scid (DNA-PK catalytic subunit deficient) fibroblasts
may show an abnormality in G1/S arrest.
Fibroblasts isolated from mouse scid lines (FS) as well as
normal mouse fibroblasts (FC), both of which contain wild-type p53,
were exposed to 8 Gy IR. The effect on cell cycle progression was
demonstrated by the accumulation of cells at the G1/S
checkpoint, which occurred at 6-9 h in FC cells, as shown in Fig.
1A. The kinetics of this
normal response has been previously described (1). However, a delayed
and attenuated G1 arrest was noted in the FS fibroblasts,
which showed little reduction in the S-phase fraction at 9 h, and
only by 24 h was there evidence of accumulation of cells in
G1 and diminution of cells in S-phase, as shown in Fig.
1B.

View larger version (15K):
[in this window]
[in a new window]
|
Fig. 1.
Flow cytometric histograms of cells prior to
irradiation (left) compared with 9 h
(center) and 24 h (right) after
irradiation. The horizontal axis of each histogram
reflects DNA content, the vertical axis represents cell
number. The upper panel shows the normal mouse embryonic
fibroblasts (FC), the center panel shows scid
cells (FS), and the lower panel shows scid cells
stably transfected with the E6 protein of human papilloma virus
(FS-E6). The rectangle is an estimate of the number of cells
in S-phase.
|
|
To demonstrate that scid cells have residual function of
p53, pXIPneoE6 was transfected and maintained in both normal and scid MEF cells. The transfected cells then expressed little
or no detectable p53 (data not shown), as has been shown by multiple previous reports. Fig. 1C shows the flow cytometric profile
in response to DNA damage in scid cells with E6 protein. At
9 h after 8 Gy of IR, there is a lack of any detectable
accumulation of cells in G1 and no decrease in the
proportion of S-phase cells. The lack of cells in G1 and
the large accumulation of cells in G2 makes the estimation
of S-phase more difficult, but the most conservative model to fit the
data supports the lack of decrease in S-phase component. At 24 h,
cells remain detectable throughout G1 and S-phase, again
suggesting the loss of any G1/S checkpoint in response to
DNA damage. The flow cytometric profile of FC-E6 in response to IR was
essentially the same as FS-E6 (data not shown). Both FS and FC cells
have been transfected with a CMV based expression vector containing a
dominant negative mutation of p53, alanine 143, and neither cell line
shows a G1/S arrest or p21WAF1/cip1 response (data
not shown) confirming that the responses are
p53-dependent.
The Rise in Level of p53 Is Normal in Scid Fibroblasts--
An
increase in p53 protein levels appears to be closely linked to the
IR-induced G1 arrest. Since scid fibroblasts
lack the normal arrest kinetics in DNA synthesis after exposure to IR, the relationship between the scid phenotype (deficient in
DNA-PK activity) and p53 function was investigated. Immunoblotting of the lysates from FS and FC cell cultures at times following exposure to
8 Gy IR is shown in Fig. 2A.
The response of p53 to DNA damage showed only a 3-fold increase at 3-6
h and was not significantly different between FS and FC lines. Although
the cell cycle arrest kinetics were delayed in the scid MEF,
the rise and fall of p53 appeared to be normal. Interestingly in
scid cells, there is a second rise in p53 levels at 24 h. These findings were reproduced in three independent experiments and
was thought to be due to persistent DNA breaks. The conclusion is that
DNA-PK does not affect the regulation of p53 levels, which is largely
determined by degradation processes.

View larger version (42K):
[in this window]
[in a new window]
|
Fig. 2.
Immunoblot of whole cell lysates from FS
(upper panel) compared with FC (lower
panel) cells. Time points are shown from immediately
prior to irradiation, then every 3 to 12 h, and finally at 24 h. Fifty micrograms of protein were loaded per lane. A, p53
using pAb421. The positive control was SAOS-2 cells with wild-type p53
transiently transfected, the negative control was SAOS-2 alone, which
contains no p53. B, p21WAF1/cip1. The positive
control was a rat embryo fibroblast after 8 Gy IR, and the lane marked
( ) was for the same cell prior to IR exposure. C, MDM2.
The positive control was SAOS-2 cells with transient transfection of
pCMV-MDM2. The 90-kDa position of MDM2 is marked with an
arrow.
|
|
p21 and MDM2 Induction Is Delayed and Attenuated in Scid
Fibroblasts--
Analysis of the same cell lysates from FS and FC for
induction of p21, revealed that the rise of p21WAF1/cip1 was
attenuated and delayed in FS cells. Fig. 2B shows a 3-fold increase in p21WAF1/cip1 in the scid MEF, FS, which
was seen maximally at 12-24 h. It is noted that the p21 band in FS
cells appears to be slightly shifted below the control band. However,
the stringency of the blot suggests a specific, albeit weak, signal. No
time points beyond 24 h were evaluated, although cells which
continue to grow after irradiation show that p21WAF1/cip1
levels return to baseline levels (data not shown). In contrast, a
>5-fold induction seen at 3-6 h in FC cells. These findings are in
keeping with the differences in cell cycle arrest kinetics previously
demonstrated in Fig. 1.
Since MDM2 is known to be induced in response to DNA damage in a
p53-dependent manner (10), the induction of MDM2 was
studied in FS cells and found to be attenuated and delayed as for
p21WAF1/cip1. Fig. 2C shows that the induction of
MDM2 shows little response at 3, 6, 9, and even 12 h after 8 Gy of
IR, but by 24 h the MDM2 level had increased 2-fold. It is noted
that MDM2 immunoblots show 2 bands at the 90 kDa size, which was
observed in their original characterization (53), but it is not known
what these two bands represent. The ratio of the two bands does not
appear to change with DNA damage in either cell type, and thus there is
nothing to link the different bands to post-translational modification. In contrast to FS, the normal FC cell showed 5-fold induction of MDM2
by 3 h, which is at least coincident with or even preceding the
p53 response. Taken together, these two gene products,
p21WAF1/cip1 and MDM2, which are known to be transactivated in
a p53-dependent manner provide evidence that the p53
response to DNA damage is not normal in scid cells with
deficient DNA-PK activity.
Nuclear Accumulation of p53 Is Abnormal in Scid Cells--
Nuclear
lysates were examined for the levels of p53, MDM2, and p21 following
exposure to IR. Fig. 3A shows
the rise and fall of nuclear p53 in FS and FC cells. As seen in whole
cell lysates, there was no deficit in the ability of scid
cells to sustain a response to IR by an increase in level of p53.
Furthermore, the level of p53 in scid cells rises by 3-6 h,
falls by 9 h, and then rises again by 24 h, in contrast with
FC cells which show little secondary rise at 24 h. However, the
most striking difference between the cells was the significantly higher
levels of p53 found in the nucleus of scid cells at all time
points (shown in Fig. 3B). The regulation of p53 between the
cytoplasm and nucleus appears to be abnormal in scid cells,
and this was confirmed by immunofluorescence (Fig.
4). Prior to irradiation, p53 was found
to be localized predominantly within the cytoplasm in normal FC cells;
following IR, p53 was found to translocate to the nucleus (as reported
previously (54, 55)). In scid cells, p53 was found at high
levels in the nucleus prior to irradiation.

View larger version (53K):
[in this window]
[in a new window]
|
Fig. 3.
p53 immunoblot of nuclear lysates, probed
with pAb421. Time points are immediately prior to irradiation and
3, 6, 9, and 24 h after irradiation. Thirty micrograms of protein
were loaded per lane. A, separate panels for FS
(upper) and FC (lower); positive control is
SAOS-2 with wild-type p53. B, FS and FC run on same gel, and
filter probed as before, to show relative levels of p53 in the
nucleus.
|
|

View larger version (25K):
[in this window]
[in a new window]
|
Fig. 4.
Immunofluorescence of FC and FS cells,
stained with pAb421, without (left) and with
(right) IR exposure. Spindle shaped fibroblasts
show predominantly cytoplasmic p53 in FC cells (upper panel)
prior to irradiation, and a predominantly nuclear staining pattern
after IR. FS cells (lower panel) show a predominantly
nuclear staining pattern with or without IR.
|
|
Nuclear MDM2 Shows Abnormal Response to IR in Scid Cells--
The
levels of MDM2 in nuclear lysates were also found to be abnormal in
scid cells compared with normal FC cells. Prior to irradiation, detectable levels of MDM2 were found in the nucleus in
both FC and FS cells, with perhaps higher basal levels seen in the FS
cells (Fig. 5). However, after exposure
to IR, the levels of MDM2 in the nucleus of FS cells decreased, before
returning to basal levels by 24 h. In contrast, the level of MDM2
in FC cells rapidly increased by 3 h after IR and gradually
returned to basal levels by 24 h. The levels of
p21WAF1/cip1 in nuclear lysates were undetectable in both cell
types.

View larger version (32K):
[in this window]
[in a new window]
|
Fig. 5.
Immunoblot of nuclear lysates, probed with
2A10 antibody for MDM2. Time points are immediately prior to
irradiation and 3, 6, 9, and 24 h after irradiation. Thirty
micrograms of protein were loaded per lane. Separate panels for FS
(upper) and FC (lower); positive control is
SAOS-2 transfected with pCMV-MDM2, a derivative of pCMV-SN3 (66), with
human MDM2 cloned into the BamHI sites. An arrow
marks the position of MDM2.
|
|
The conclusion, from these observations, is that the regulation of p53
between the nucleus and cytoplasm is abnormal in cells deficient in
DNA-PK activity, and the consequence of this abnormal regulation is
impaired activation of downstream genes p21WAF1/cip1 and MDM2.
Furthermore, the delayed and attenuated G1/S cell cycle checkpoint appears to be a consequence of the effect on
p21WAF1/cip1. MDM2 appears to decrease from the nucleus after
DNA damage when there is a lack of DNA-PK activity, but the opposite
response is seen in normal cells.
 |
DISCUSSION |
The results in this paper suggest that, in scid cells,
activation of downstream genes by p53, in response to DNA damage, is delayed and attenuated, and only by looking at a detailed time course
is this effect seen for both induced protein levels and G1/S cell cycle arrest. They further suggest that, even
without exogenous DNA damage, p53 is accumulating in the nucleus, but in a form that is less capable of triggering the downstream effects. Both of these observations are consistent with the hypothesis that
DNA-PK is modifying p53 function, directly or indirectly, without
affecting the ability of cells to accumulate p53 protein.
Altered p53-dependent DNA Damage Response in Scid
Cells--
The signaling pathway regulating the degradation of p53 in
response to DNA damage, which is thought to be the main mechanism regulating the levels of p53 following DNA damage (56, 57), is not
critically dependent upon DNA-PK. We and others (34, 35, 38) have found
that the levels of p53 rise and fall in scid mouse derived
MEF just the same as in normal MEF. However, the ability of p53 to
activate downstream genes appears to be, at least in part, dependent on
DNA-PK, consistent with and complementary to the findings of Woo
et al. (39). A link between DNA-PK and p53, using an
in vivo response has not previously been reported. The
justification for this conclusion is the kinetics of the induction of
the downstream genes p21WAF1/cip1 and MDM2, and the kinetics of
G1/S arrest. The distribution of p53 between the cytoplasm
and nucleus appears to be the major defect: abnormally high levels of
p53 are seen in the nucleus in scid cells but this p53 is in
a form that is relatively inactive and less able to activate downstream genes.
This abnormal G1/S arrest appears to be in contradiction to
recent reports for MEF (34, 38), which demonstrated that the G1/S arrest in response to DNA damage in scid
cells was equivalent in magnitude to that of the wild-type parental
line. However, the report by Huang et al. (34) shows
G1/S arrest profiles only at 24 h, and at that time
point, little difference between normal and scid cells was
observed in our studies (see Fig. 1). The report by Rathmell et
al. (38) shows flow cytometric profiles with 5-bromodeoxyuridine
antibody analysis at 6-8 h following 8 Gy IR in MEF, which appears to
be equivalent to the analysis presented in this report. However, it is
clear that both normal and scid cells have a significant
tetraploid population, which is a sign of genetic instability (58). In
our unpublished observations with MEF, when tetraploidy is observed,
loss of p53 function follows within a small number of further passages
in culture. As a consequence, MEF showing tetraploidy are rejected for
further use and analysis.
Cells deficient in DNA-PK activity by mutation or knockout of Ku 80 have been reported to have a normal G1/S checkpoint at time
points less than 12 h (59). It is not clear whether this reflects
functional differences between scid, which has mutant DNA-PKcs, and Ku 80 null cells. A DNA-PKcs-null mouse has recently been
established (60, 61) but p53 responses have not yet been reported for
comparison. It was of considerable interest that the DNA-PKcs null
mouse and the MEF cells derived from the mouse showed a phenotype that
was identical to the scid mouse and scid cells,
in all aspects in relation to DNA repair and V(D)J recombination, suggesting that the phenotype of scid is much closer to the
null phenotype, despite the residual detectable DNA-PKcs protein and DNA-PK activity in the in vitro assay (39). The relationship between the measured DNA-PK activity in vitro and the
phenotype seen in vivo may not correlate as well as
predicted by purely in vitro studies. At the time of our
studies, there was no alternative DNA-PKcs deficient line to study for
comparison. The hamster line, V3 (62), which does have a mutation in
DNA-PKcs, has inactived p53.
It is only by analyzing cell cycle profiles, induced levels of
p21WAF1/cip1 and MDM2 at every 3 h, that the differences
between scid cell and normal cells becomes apparent.
Rathmell et al. (38) show a Northern blot analysis of p21
mRNA, at a single time point 4 h after exposure to IR, and
report equivalent induction. A single time point could reflect a
decreasing value for normal cells and an increasing value for
scid cells. No protein analysis, in conjunction with the
mRNA studies, was shown for p21, perhaps because the levels are
extremely low (as we observed). Analysis of cells with p53 function
eliminated would show that the p21 induction was p53-dependent (63). The elevation of p21 and MDM2 levels
beyond 12 h shown in this report suggests that their activation by
p53 is delayed and attenuated. One feature of scid cells is
that the levels of p53 peak again at 24 h, and this is presumably
due to unrepaired damage continuing to maintain the signal to inhibit degradation. It is clear that residual p53 function is present in
scid cells, as elimination of p53 either by knockout or by E6 transfection results in complete loss of the G1/S
checkpoint and a failure to induce p21 or MDM2.
DNA-PK, p53, and MDM2--
Regulation of the function of p53 as a
transcription factor is dependent on an autoregulatory feedback loop
with MDM2 (5). It has been shown that DNA-PK phosphorylates p53
in vitro at serines 15 and 37 (17), and that various types
of DNA damage result in an increase of phosphorylated p53 at serine 15 (18). When p53 is phosphorylated at this location, its ability to
transactivate is improved and its interaction with MDM2 is inhibited.
In principle, this would appear to predict a model in which deficient
DNA-PK activity results in unphosphorylated p53 at serines 15 and 37, which in turn would result in both decreased immediate transactivation and slower stimulation of downstream genes. These predictions are
entirely in keeping with our observations. In addition, Fiscella et al. (20) reported that a cell line containing a serine 15 phosphorylation mutant of p53 had a delayed G1/S
checkpoint. However, Shieh et al. (18) also report (without
showing specific data) that p53 is constitutively phosphorylated at
serine 15 in scid cells, which is not the prediction of the
model. It may be that focusing on one phosphorylation site is an
oversimplification of the constitutive regulation of p53 and its
response to DNA damage. The modification of other sites within p53 may
affect the impact of phosphorylation in the N-terminal domain. Another possibility is that MDM2 is also phosphorylated by DNA-PK, and regulation of MDM2 function is also controlled by phosphorylation or
dephosphorylation. The amino acid sequence of MDM2 does predict potential substrate sites for DNA-PK.
Alterations of p53 in scid without DNA Damage: Nuclear Cytoplasmic
Distribution--
Further characterization of the activation of p53
and the transactivation of downstream genes has revealed abnormalities
in the localization of p53 between the cytoplasm and nucleus, from both
immunoblotting and immunofluorescence. Curiously, there were high
levels of p53 in the nucleus of scid cells prior to any
exposure to IR, whereas normal cells maintain p53 predominantly within the cytoplasm until DNA damage, when it promptly moves to the nucleus
(43). Why should there be abnormally high levels of p53 in the nucleus
in scid cells prior to irradiation? It is known that many
types of mutant p53 can accumulate in cells, with a predilection for
the nucleus, but in scid cells it was shown that partial p53
function is preserved. Perhaps the accumulation of p53 into the nucleus
is a reflection of a repair deficient cell, which has chronically
accumulated DNA damage, which in turn maintains a damage signal to p53.
If there were a physiologic accumulation of p53, then transactivation
should not be impaired and the cell should either be growth arrested or
ready to arrest with little delay.
Loss of DNA-PK activity in scid cells results in a loss
normal maintenance of p53, as well as an attenuated and delayed DNA damage response. We expect the DNA-dependent protein kinase
to be a DNA damage-dependent protein kinase, but activity
of the kinase can be detected in the absence of exogenous DNA damage and in a cell cycle-dependent manner, with peaks in
G1 and G2 (64). It would clearly be undesirable
to respond to the DNA breaks in S-phase as if they were exogenous DNA
damage, and part of normal maintenance may keep p53 in the
appropriately modified state for the different phases of the cell
cycle. The phosphorylation state of a protein is the predominant
mechanism for regulating transport between the cytoplasm and nucleus.
Although p53 accumulates in the nucleus of scid cells, it
does not allow rapid transactivation, again implying that p53 in scid
cells has a different post-translational modification from normal cells.
The Functional Significance of Delayed Transactivation--
The
loss of DNA-PK activity in scid cells results in decreased
double-strand break repair, as a consequence of impaired non-homologous recombination, and an apparent direct role of DNA-PK in DNA repair. It
seems likely that DNA-PK may also play a role in sensing DNA damage,
and as a protein kinase, function in a signal transduction pathway.
What functional consequences occur as a result of delayed transactivation? It is not clear how this contributes to the phenotype of scid cells, whose defect seems to be largely explained by
the impairment of non-homologous recombination. Furthermore,
scid cells (in contrast with cells derived from patients
with ataxia telangiectasia) are not hyper-mutable which could result
from a loss or impairment of the G1/S
checkpoint.2 Cells deficient
in p21WAF1/cip1 which have a partial loss of the
G1/S checkpoint, also have no significant phenotype other
than the checkpoint abnormality (65).
The delayed transactivation could potentially affect the activation of
apoptotic pathways, but this has not been evaluated in this paper or
other studies. MEF are not a useful cell system to study apoptosis.
DNA-PK mutations are not generally found in genetically unstable cells
or in transformed or tumor cells. Therefore, although abnormalities of
p53 activation have been detected in scid cells, the
functional consequences of these abnormalities have not been
established: it is clear that many functions of p53 remain intact
within scid cells. It is likely that many signal pathways
may contribute to post-translational modification of p53: loss of
DNA-PK activity may cause an imbalance in the normal regulatory
process. The combination of delayed G1/S checkpoint, abnormal p21WAF1/cip1, and MDM2 induction, and abnormal nuclear
localization of p53 and MDM2 provide compelling evidence that DNA-PK
activity does affect p53 function. The additional recent evidence about
the effect of DNA damage, resulting in phosphorylation of serines 15 and 37, activated DNA binding and increased transactivation in a
reporter assay supports this view. The signal pathways from DNA damage
to p53 appear to be distinct between post-translational modification of
p53 (which is DNA-PK dependent) and degradation of p53 (which thus far
appears independent of DNA-PK).