From the Institute of Molecular Medicine and Genetics, Program in Gene Regulation, Medical College of Georgia, Augusta, Georgia 30912
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ABSTRACT |
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Purified heat shock transcription factor 1 (HSF1) binds to both the regulatory and catalytic components of the
DNA-dependent protein kinase (DNA-PK). This observation
suggests that DNA-PK may have a physiological role in the heat shock
response. To investigate this possibility, we performed a comparison of
cell lines that were deficient in either the Ku protein or the DNA-PK
catalytic subunit versus the same cell lines that had been
rescued by the introduction of a functional gene. DNA-PK-negative cell
lines were up to 10-fold more sensitive to heat-induced apoptosis than matched DNA-PK-positive cell lines. There may be a regulatory interaction between DNA-PK and HSF1 in vivo, because
constitutive overexpression of HSF1 sensitized the DNA-PK-positive
cells to heat but had no effect in DNA-PK-negative cells. The initial
burst of hsp70 mRNA expression was similar in DNA-PK-negative and
-positive cell lines, but the DNA-PK-negative cells showed an
attenuated rate of mRNA synthesis at later times and, in some
cases, lower heat shock protein expression. These findings provide
evidence for an antiapoptotic function of DNA-PK that is experimentally separable from its mechanical role in DNA double strand break repair.
The DNA-dependent protein kinase
(DNA-PK)1 is composed of a
470-kDa catalytic subunit (DNA-PKcs) and a 70/80-kDa heterodimeric regulatory component known as Ku protein. Ku protein binds avidly to
DNA ends (1) and recruits DNA-PKcs to form an active complex (2-4).
Mutations in DNA-PKcs or Ku protein have been described in mammals,
flies, and yeast, and in each case, the mutant organism is deficient in
double strand break repair, sensitive to ionizing radiation, or both
(reviewed in Refs. 5 and 6). Binding of DNA-PK components to DNA is
believed to be the first step in a repair pathway that involves a
number of other gene products (reviewed in Ref. 7).
It is not clear if the ability of DNA-PK to protect against the
cytotoxic effect of ionizing radiation is attributable solely to the
mechanical role of the enzyme in DNA repair or whether DNA-PK also
participates in antiapoptotic signaling. There have been several
reports that DNA-PK physically interacts with signaling molecules,
including poly(A)DP-ribose polymerase, I In the present study, we investigate the in vivo role of a
previously described interaction between DNA-PK and the heat shock transcription factor, HSF1 (12, 13). These prior studies have shown
that purified HSF1 binds to both the Ku protein and DNA-PKcs (13). The
binding requires a phylogenetically conserved region of HSF1 that
includes amino acids 203-280 and results in a stimulation of DNA-PK
phosphorylation activity of up to 20-fold in an in vitro reaction (13). These observations suggest that DNA-PK may be involved
in regulating some aspect of the heat shock response in
vivo.
Other evidence also suggests an involvement of DNA-PK in the
response to heat. Overexpression of human Ku70 in rat cells decreases expression of heat shock protein 70 (hsp70) (14, 15). It was originally
proposed that this might reflect competition of HSF1 and Ku protein for
binding to heat shock promoter elements (16), although this mechanism
has not been supported by later studies (17). Even if the underlying
mechanism is unclear, the finding that ectopic expression of a Ku
protein subunit perturbs expression of a heat shock protein supports
the idea that DNA-PK participates in the heat shock response.
It is also known that heat treatment potentiates the cytotoxic effect
of ionizing radiation, a phenomenon called thermal radiosensitization (18-20). Interestingly, some cell lines deficient in Ku protein or
DNA-PKcs do not show thermal radiosensitization (i.e. their intrinsically high sensitivity to radiation is not further increased by
heat) (21, 22). This implicates DNA-PK in the thermal
radiosensitization phenomenon. Ku protein may be intrinsically
heat-labile (23, 24), in which case heat treatment may reduce double
strand break repair. An alternative possibility is that binding of
DNA-PK to HSF1 in heat-treated cells competes with its ability to
interact with the DNA repair machinery.
In the present work, we show that DNA-PK protects against heat-induced
apoptosis. Protection is abrogated by introduction of a constitutively
expressed HSF1 gene. These studies provide evidence for an
antiapoptotic function of DNA-PK that is separable from its role in DNA
repair. We find that there are some differences in the time course of
induction of hsp70 mRNA and protein in DNA-PK-positive and
DNA-PK-negative cells. It is possible that a difference in the state of
the transcriptional machinery accounts for the differential heat
sensitivity. Alternatively, protection from apoptosis may occur by a
nontranscriptional mechanism.
Cell Culture, Clonogenic Survival Assays, and Generation of
Stably Transfected Cell Lines--
For cell culture and clonogenic
assays, HeLa, scva2, sc(8)-10, xrs-6cvec, xrs-6cKu80, and AA8 cells
were grown at 37 °C with 5% CO2 in minimal essential
medium (Life Technologies, Inc.), with 10% heat-inactivated fetal
bovine serum, nonessential amino acids, and 50 µg/ml gentamycin.
Medium for sc(8)-10, xrs-6cvec, and xrs-6cKu80 cells contained 400 µg/ml Geneticin. For clonogenic survival assays, cells were
heat-treated as indicated in the figure legends and counted. Equal
numbers of cells were plated and allowed to grow for 7 days. Colonies
were fixed in 5% glutaraldehyde and stained with crystal violet, and
colonies of more than 20 cells were counted.
For transfection, 107 cells were electroporated in 800 µl
of minimal essential medium with 20 µg of expression plasmid and 5 µg of pBabe plasmid, which contains a puromycin selection marker (25). scva2 and sc(8)-10 cells were electroporated at 300 V, 1500 microfarads. xrs-6cKu80 and xrs-6cVec cells were electroporated at 300 V, 2500 microfarads. After transfection, cells were plated in minimal
essential medium (without Geneticin). After 24 h, Geneticin (400 µg/ml) and puromycin (3 µg/ml for scva2 and sc(8)-10, 5 µg/ml for
xrs-6cVec and xrs-6cKu80 cells) were added.
Detection of Apoptosis by Enzyme-linked Immunosorbent Assay and
dUTP Labeling--
Apoptosis was quantitated by measuring release of
histone-DNA fragments into the cytoplasm using a commercial assay
(Roche Molecular Biochemicals). Briefly, cytoplasmic extracts
corresponding to 103 cells were incubated for 2 h in
microtiter wells that had been precoated with anti-histone mouse
monoclonal antibody (clone H11-4). The wells were washed, and mouse
anti-DNA peroxidase-conjugated monoclonal antibody (clone MCA-33) was
added. Incubation was continued for 2 h, and the wells were
developed with ABTS substrate. Signal was read as the difference
between A405 and
A492.
For immunofluorescence, cells were plated on glass coverslips and
allowed to grow for 24 h and then heat-treated as indicated in the
figure legends. Cells were fixed in 4% paraformaldehyde in
phosphate-buffered saline (PBS) for 30 min at room temperature, washed,
and incubated for 2 min on ice with a solution containing 0.1% Triton
X-100 and 0.1% sodium citrate. Apoptotic death was scored using the
TUNEL (TdT-mediated dUTP nick-end labeling) method using
fluorescein-dUTP (Roche Molecular Biochemicals) (26). The cells were
washed and incubated for 30 min at room temperature with blocking
solution containing 5% nonfat dry milk, 0.9% NaCl, 10 mM
Tris-HCl, pH 7.2, 0.1% Tween 20, and 0.2% sodium azide. Cells were
stained for 1 h each with 1 µg of primary and Texas Red-conjugated secondary antibodies as specified in the figure legends.
Cells were washed in PBS containing 1 µg/ml diamidino 2-phenylindole
dihydrochloride hydrate (DAPI) (Aldrich) and mounted using 5 µl of
mounting medium containing 10% Mowiol (Hoechst), 25% glycerol in 200 mM Tris-HCl, pH 8.5.
Immunoblotting--
To obtain cell extracts, 5-10 × 106 cells were heat-treated as described in the figure
legends, washed in PBS, scraped from the plate, suspended in 200 µl
of PBS, and lysed by the addition of 200 µl of 2× SDS-polyacrylamide
gel electrophoresis sample buffer. Equal quantities of protein from
each extract were fractionated by SDS-polyacrylamide gel
electrophoresis. The proteins were transferred to a polyvinylidene
difluoride or nitrocellulose membrane by electroblotting in a buffer
containing 25 mM Tris base, 192 mM glycine, and
20% methanol. The membrane was blocked in PBS containing 0.1% Triton X-100 and 5% nonfat dry milk and sequentially probed with the primary
antibody for 2 h, washed with PBS containing 0.1% Triton X-100,
probed with alkaline phosphatase-conjugated secondary antibody, and
finally incubated for 5 min with enzyme-catalyzed fluorescence substrate (Vistra ECF Substrate, Amersham Pharmacia Biotech) or 5-bromo-4-chloro-3-indolyl phosphate/nitro blue tetrazolium substrate (Promega). The membrane was dried, and signal was quantitated with a
Storm imaging system (Molecular Dynamics, Inc., Sunnyvale, CA).
Electrophoretic Mobility Shift Assay--
Electrophoretic
mobility shift assay was performed as described previously (27).
Briefly, 5 × 106 cells were heat-treated as indicated
in the figure legends, washed with PBS, and lysed in 200 µl of buffer
containing 10 mM Pipes, pH 7.9, 0.4 M NaCl, 0.1 mM EGTA, 0.5 mM dithiothreitol, 5% glycerol, and 0.5 mM phenylmethylsulfonyl fluoride. For binding, 20 µg (approximately 10 µl) of cell extract was mixed with 10 µg of
tRNA, 1 µg of sheared Escherichia coli DNA, 10 µg of
poly(dI-dC), and 1 ng of [ Nuclear Run-on and Northern Blot Analysis of RNA
Synthesis--
Total RNA was isolated from untreated or heat-treated
HeLa, scva2, or sc(8)-10 cells by the guanidine isothiocyanate method, fractionated by formaldehyde-agarose electrophoresis, and transferred to a nitrocellulose membrane as described (28). The filter was probed
with a cDNA corresponding to the complete sequence of human hsp70D
(kindly provided by Dr. Richard I. Morimoto).
Nuclear run-on assays were performed as described (29) with
modifications. Nuclei were prepared from 5 × 106
cells that were heat-treated as described in the figure legends. Nuclei
that had been stored in 200 µl of glycerol storage buffer (50 mM Tris-HCl, pH 7.4, 10 mM NaCl, 3 mM MgCl2, and 0.5% Nonidet P-40) were mixed
with 200 µl of 2× reaction buffer (10 mM Tris- HCl, pH
8.0, 5 mM MgCl2, 0.3 M KCl, 10 mM dithiothreitol, 1 mM concentration each of
ATP, GTP, and UTP), and 10 ml of [
To prepare membranes for hybridization with the radiolabeled
run-on RNA, 20 µg of plasmid DNA were digested with restriction endonucleases. Plasmid containing human DNA-PK-deficient Cells Have an Increased Sensitivity to
Heat--
To test whether the heat shock response is abnormal in
DNA-PK-deficient cells, we made use of mutant cell lines that lack either DNA-PKcs or Ku80, as listed in Table
I. The DNA-PKcs-negative cell line,
scva2, derived from a severe combined immunodeficiency (scid) mouse
(31), is homozygous for an allele of DNA-PKcs that has a nonsense
mutation, causing a small truncation at the C terminus of the protein
(32-34). This results in loss of kinase activity and decreased levels
of antigenically reactive DNA-PKcs (35). A matched DNA-PKcs-positive
cell line, sc(8)-10, was derived from scva2 by introduction of a
centromeric fragment of human chromosome 8 containing the DNA-PKcs
structural gene, which rescues radiation resistance and DNA-PK activity
(35). The Ku80-negative cell line is derived from xrs-6 (36), a Chinese
hamster ovary cell line that has a mutation in the Ku80 structural gene
that affects RNA splicing and abolishes Ku80 protein expression (37).
The xrs-6cKu80 line was derived from xrs-6 by transfection with a human
Ku80 cDNA, which rescues Ku protein expression and radiation resistance, and the isogenic xrs-6cvec line was derived by transfection with the same expression vector lacking the Ku80 cDNA (38). The
unmutated, Ku80-positive, AA8 CHO cell line was also used in some
experiments.
Cells were subjected to heat treatment for various times at 43 °C
and tested for reproductive viability, i.e. the ability to
form colonies in a monolayer cloning assay. The surviving fraction was
calculated by counting the number of colonies formed by the heated
cells and dividing by the number of colonies formed by unheated control
cells plated at the same time. The data reveal a substantial difference
in heat sensitivity (Fig. 1). The
DNA-PKcs- and Ku80-negative cells (sva2, xrs-6cvec) are up to 30-fold
more sensitive to heat-induced cell death than their genetically
matched counterparts (sc(8)-10, xrs-6cKu80). The Ku80-negative cells
(xrs-6cvec) were also more heat-sensitive than the unmutated AA8
Chinese hamster ovary cell line, although the difference was not as
great, perhaps reflecting a level of endogenous Ku protein in AA8 cells
that is somewhat lower than in the rescued xrs-6cKu80 cell line.
Differences in cell survival were seen at all heat doses. The observed
differences in heat sensitivity are of the same magnitude as the
differences in ionizing radiation sensitivity previously reported with
these cell lines (31, 38). These data indicate that DNA-PK provides substantial protection against heat-induced cell killing under the
conditions of these experiments and that both the Ku and DNA-PKcs components are required.
Heat Sensitivity Reflects Increased Apoptosis in DNA-PK-deficient
Cells--
Heat-induced cell death can occur by either apoptosis or
necrosis, depending on the heat dose and the cell type. At temperatures ranging between 42 and 45 °C, many types of cells die by apoptosis. At higher temperatures, necrosis is the prevalent form of cell death
(39-46).
To determine whether the DNA-PK-deficient cells in our experiments were
undergoing cell death by an apoptotic mechanism, we used a quantitative
immunoassay to detect release of histone-DNA complexes into the
cytoplasm, which is diagnostic of apoptotic death (47). Heat treatment
resulted in an increase in apoptotic complexes in all cell lines. The
increase was greater, however, in the DNA-PKcs-negative and
Ku80-negative cells, as compared with the matched controls (Fig.
2). For all cell lines, the level of
apoptotic histone-DNA complexes reached a maximum within 4 h after
heat treatment. This rapid onset of cell death is consistent with
results of previous studies using heat-treated cells (45, 46).
The difference in the amount of apoptotic histone-DNA complexes
measured in DNA-PK-positive and -negative cell populations was on the
order of 2-fold. This is a smaller difference than what was seen in the
monolayer cloning assay. It is likely that the immunoassay
underestimates the differences between cell lines because it measures
only complexes recovered from intact cells that remain attached to the
culture flask. Many of the DNA-PK-deficient cells disintegrated or
detached from the flask prior to harvesting, reducing the amount of
apoptotic complexes that were detected.
We also used a different assay that allowed us to visualize apoptotic
cells individually and thus measure the fraction of apoptotic cells
that were present in the population at a given time. In this assay,
fluorescein-labeled dUTP was incorporated into the ends of apoptotic
DNA fragments by terminal deoxynucleotidyl transferase and visualized
by fluorescence microscopy. The same samples were also stained with the
DNA binding dye DAPI to allow visualization of all cells present in the preparation.
Heat treatment of DNA-PKcs-negative cells resulted in a marked increase
in the fraction of fluorescein-labeled cells, as compared with the
unheated control population (Fig. 3).
Quantitation showed that the fraction of apoptotic cells in the
DNA-PKcs-negative population increased more than 10-fold, whereas there
was little change in the fraction of apoptotic cells in the
DNA-PKcs-positive population (Fig. 3B). Similar results were
obtained in experiments using Ku80-negative and -positive cells and
will be presented below.
The difference in cell death measured by the immunofluorescence assay
(about 10% in scva2 cells) compared with the colony forming assays
(99% under similar conditions) may be in part attributed to inherent
underestimation by the immunofluorescence method, because only cells
that are undergoing apoptosis at a single time point are scored.
Some apoptotic cells may float away before they can be fixed and
detected, and others may be destined to undergo apoptosis at later
times and also escape detection. By contrast, the colony forming assay
provides a measure of cumulative death in the entire population. In
addition, the colony formation assay scores cells that do not undergo
apoptosis but nevertheless lose reproductive viability.
Overexpression of HSF1 Constructs in xrs-6cKu80 Cells Results in
Increased Apoptosis--
The preceding experiments establish that
DNA-PK protects cells against heat-induced apoptosis. We wished to
determine whether this protection was modulated by HSF1. Previous
studies have shown that HSF1 binds to and regulates DNA-PK in
vitro (12, 13). If overexpression of wild-type or mutant HSF1
could be shown to affect DNA-PK-mediated protection against
heat-induced apoptosis, this would be evidence for a regulatory
interaction between these proteins in vivo. Accordingly, we
established populations of transfected cells expressing HSF1
derivatives in a Ku80-positive and -negative background.
A map of the HSF1 derivatives used in this experiment is shown in Fig.
4A. All of these derivatives
form trimers and bind to DNA in vitro (13). They differ in
their ability to interact with DNA-PK. HSF1-(1-450), which contains
the first 450 of the 529 amino acids of human HSF1, binds strongly to
DNA-PK components in vitro and stimulates DNA-PK activity
(13). HSF1
Plasmids expressing each of these constructs, a plasmid expressing
full-length HSF1, as well as the pcDNA3 vector alone, were cotransfected with a puromycin selection marker into Ku80-positive and
-negative cells. Populations were established and tested for HSF1
expression by immunofluorescence. As shown in Fig. 4B,
xrs-6cKu80 cells transfected with pcDNA3 alone expressed a low
level of endogenous HSF1. Cells transfected with expression constructs
expressed higher levels of HSF1. As expected, the endogenous HSF1 was
predominantly cytoplasmic in unheated control cells and partially
relocalized to the nucleus after heat treatment. The HSF1
Heat shock-induced apoptosis was measured in each transfected
population using the fluorescein-dUTP incorporation assay.
Representative data are shown in Fig.
5A, with quantitation of these
and other experiments in Fig. 5, B and C. The
Ku80-positive cells transfected with pcDNA3 vector alone
(xrs-6cKu80 pcDNA3) were relatively heat-resistant, as expected
(Fig. 5A, top row). The same cells
transfected with the HSF1-(1-450) showed an increase in heat
sensitivity of about 8-fold (Fig. 5A, second
row). The Ku80-negative cells transfected with pcDNA3
vector alone (xrs-6cvec pcDNA3) were about 20-fold more sensitive
to heat than the Ku80-positive cells (Fig. 5A, third row). Interestingly, transfection of these
cells with HSF1-(1-450) produced no change in heat sensitivity (Fig.
5A, fourth row). That is, constitutive
overexpression of HSF1 under the conditions of this experiment
specifically suppresses the protective effect of DNA-PK. The simplest
interpretation is that HSF1 is a negative regulator of DNA-PK in
vivo. The implications of this finding will be considered further
under "Discussion."
As a further test of the specificity of the interaction between HSF1
and DNA-PK, we evaluated cells that had been transfected with mutant
HSF1 derivatives. Expression of HSF1
It is of interest that, while some of the HSF1 derivatives sensitized
cells to heat, neither the full-length HSF1 nor any of the HSF1
derivatives exhibited a protective effect. This finding must be
considered in the context of the absence of a functional inducible
hsp70 gene in these cells (see "Discussion").
Expression of Heat Shock Genes in DNA-PKcs- and Ku-deficient
Cells--
Heat shock initiates a well characterized program of gene
expression in mammalian cells. HSF1 plays a central role in this process. Heat treatment induces the conversion of HSF1 from a latent to
an active form, which is followed by binding of HSF1 to promoters of
heat shock genes, transcription of heat shock mRNAs, and synthesis
of heat shock proteins. Because of the previous experiments showing
that there is a regulatory interaction between DNA-PK and HSF1, it was
of interest to examine various steps in this pathway in DNA-PK-positive
and -negative cell lines.
Electrophoretic mobility shift assays were performed with extracts of
various cell lines in order to detect HSF1 DNA binding activity (Fig.
6A). All of the cell lines
showed induction of binding activity upon heat treatment for 20 min at
43 °C, as evidenced by formation of a characteristic HSF1-DNA
complex. In each case, the complex was specific for the heat shock
element sequence, as demonstrated by its sensitivity to competition
with a nonradiolabeled oligonucleotide. These data show that Ku and
DNA-PKcs are not required for conversion of endogenous HSF1 to the
DNA-binding form.
The induction of mRNA for the major inducible heat shock protein,
hsp70, was examined in detail in DNA-PKcs-positive and -negative cell
lines. To assess the rate of hsp70 gene transcription, we performed
nuclear run-on assays. Nuclei were isolated, run-on RNA was synthesized
in the presence of [
We also measured hybridization of run-on RNA to a cDNA encoding
another heat shock protein, hsp27 (49), and to a cDNA encoding a
housekeeping protein,
Steady state hsp70 mRNA levels were measured by Northern blot
analysis (Fig. 6C). Heat shock reduced the yield of total
RNA from DNA-PK-negative cells, and in order to compensate, we loaded an equal mass of RNA, rather than RNA from an equal number of cells, in
each lane. Under these conditions, the amount of hsp70 transcript was
essentially identical in DNA-PKcs-positive and -negative cell lines.
Hybridization to
To determine whether there was a defect in production of heat shock
proteins in DNA-PK-positive and -negative cells, we performed immunoblotting. The major inducible form of hsp70 (hsp72) was detected
in both DNA-PKcs-positive and -negative cell lines (Fig. 7A). Induction in the
DNA-PKcs-negative cells was somewhat delayed, however, and reached a
3-4-fold lower steady state level. To examine the expression of hsp70
at the individual cell level, we performed immunofluorescence assays.
The level of hsp70 was uniformly reduced throughout the
DNA-PKcs-negative population (Fig. 7B). Overlay of the
anti-hsp staining with fluorescein/dUTP staining showed no apparent
correlation between the level of hsp70 proteins and the probability of
apoptotic DNA fragmentation in individual cells (data not shown).
Taken together, the results of these experiments suggest that the
initial steps in the induction of heat shock transcriptional response,
including the activation of HSF1 DNA binding and the onset of hsp70
mRNA synthesis, occur normally in DNA-PKcs-negative cells. There is
an attenuation of the later steps of the heat shock response, including
a reduction in the rate of hsp70 transcription after 2 h of
recovery and in levels of translated hsp70 protein. These later effects
could be secondary, however, and attributable to the imminent onset of
cell death in the DNA-PKcs-negative population.
Experiments were also performed to measure the induction of hsp70
mRNA and protein in xrs-6c-derived cell lines. Surprisingly, there
was no detectable synthesis of the major inducible hsp70 mRNA or
protein in either the xrs-6cvec or the xrs-6cKu80 cell line (data not
shown). This apparent transcriptional silencing may be attributable to
a secondary mutation in the xrs-6 cells, which were originally derived
from a chemically mutagenized population (36), or it may be some other
clonotypic effect. Silencing of the major inducible form of hsp70 has
been previously described in several murine cell lines, and it is
attributable in some cases to promoter methylation (52, 53).
Immunoblotting with a broad specificity polyclonal antibody showed that
the hsp73 heat shock cognate protein was induced by heat in the
xrs-6cvec and the xrs-6cKu80 cell lines (not shown), indicating that
the overall heat shock transcription response is operative, although
the major hsp70 gene is silenced.
In this report, we present evidence that DNA-PK protects cells
against heat-induced cell death. Under the conditions of our experiments, this protective effect ranged up to 30-fold, depending on
the type of assay. Protection was detected using three different assays
of cell death, which measured reproductive viability, the release of
apoptotic histone-DNA complexes, and apoptotic DNA fragmentation,
respectively. The effect of DNA-PK on survival after heat shock is
similar in magnitude to the effect on survival after exposure to
ionizing radiation. This surprising result suggests that the effect of
DNA-PK on cell survival may be separable, under some conditions, from
its mechanical role in DNA double strand break repair.
It is unlikely that the heat doses used in our experiments induce
significant DNA damage. Previous work has suggested that mild heat
treatment does not induce DNA damage in mammalian cells (54, 55).
Moreover, immunoblotting of xrs-6c derivatives (not shown) indicates
that heat shock is not accompanied by induction of p53. This argues
that significant DNA damage is not present. Mutants of
Saccharomyces cerevisiae that lack Ku protein arrest at cell
cycle checkpoints when grown at a nonpermissive temperature (56). This
growth arrest is accompanied by accumulation of DNA damage-inducible
genes, however, and thus may not be comparable with the heat
sensitivity seen in mammalian cells that lack Ku or DNA-PKcs.
The finding that Ku-deficient and DNA-PKcs-deficient cells have similar
phenotypes, together with the knowledge that these proteins cooperate
biochemically, makes it plausible to assume that both components of
DNA-PK work together to establish resistance to heat-induced apoptosis.
A formal genetic test to distinguish whether Ku protein and DNA-PKcs
work together, rather than independently, will require analysis of
double mutants, which are not yet available in the mammalian system. We
have also not yet explored whether overexpression of DNA-PK components
in wild-type cells would further increase resistance to heat-induced
apoptosis. It is of interest to consider, however, that HeLa cells,
which are a rich source of DNA-PK for biochemical purification, are
highly resistant to heat-induced apoptosis, even at temperatures as
high as 45 °C.2
Experiments that measure cell death as their end point do not
distinguish whether the effect of DNA-PK is specific to heat or
reflects a broader antiapoptotic function. Other data suggest, however,
that the protective effect of DNA-PK relates in some way to its
physical interaction with HSF1. This interaction has been demonstrated
previously in vitro, using purified proteins (13). The
interaction requires an HSF1 sequence defined, in part, by the HSF1
The loss of heat resistance in xrs-6 Chinese hamster ovary cell
derivatives that overexpress HSF1 contrasts with a previously reported
increase in heat resistance in mouse cells that overexpress HSF1 (57).
This may reflect differences in the cell lines that were used.
Transcriptional silencing at the endogenous hsp70 locus in xrs-6
derivatives precludes confounding effects mediated by HSF1-induced
changes in hsp70 expression. Moreover, technical differences may have
influenced the outcome of the experiments. In particular, the present
work measured the fraction of the population in which rapid onset of
apoptotic death occurred after mild heat doses. The other work measured
loss of reproductive viability, which may occur by both apoptotic and
nonapoptotic mechanisms, and found differences between transfected and
control cells primarily at higher heat doses.
Although our results suggest that there is an interaction between HSF1
and DNA-PK, they do not inherently distinguish whether HSF1 modulates
DNA-PK activity, whether DNA-PK modulates HSF1 activity, or both. We
shall consider first the model that HSF1 regulates DNA-PK. There is
evidence that such regulation occurs in vitro: purified HSF1
stimulates DNA-PK phosphorylation activity (12, 13). The in
vivo data argue for an effect in the opposite direction, however.
Overexpression of HSF1 suppresses the ability of DNA-PK to protect
against apoptosis. It may be that binding of HSF1 in vivo
hinders the ability of DNA-PK to interact with critical target proteins
or sequesters DNA-PK in a location where it is not able to perform its
antiapoptotic function. Also, prolonged stimulation of DNA-PK might
deplete intracellular pools of active enzyme by an autophosphorylation
mechanism. Previous work has established that DNA-PK can be negatively
regulated by autophosphorylation (58). At present, we do
not know if suppression of the DNA-PK protective effect reflects
a natural mode of regulation or is a consequence of HSF1 overexpression.
An alternative model is that DNA-PK modulates HSF1 activity. A primary
function of HSF1 is to activate synthesis of heat shock mRNAs. It
appears that we can rule out a direct role of DNA-PK as a mediator of
HSF1 transcriptional activation. The interaction between HSF1 and
DNA-PK does not occur through the transcriptional activation domain
(13). Moreover, the initial burst of heat shock mRNA synthesis
occurs normally in DNA-PKcs-deficient cells. DNA-PK could have more
subtle effects on HSF1 activity, however. HSF1 exists in an equilibrium
between active and inactive forms. Phosphorylation of HSF1 at serines
303 and 307 shifts the equilibrium toward the inactive form (59-61).
This phosphorylation appears to be mediated, in part, by glycogen
synthase kinase and members of the MAP kinase family (61-63). DNA-PK
phosphorylates HSF1 at multiple sites throughout the C-terminal portion
of the protein, at least some of which are distinct from the sites that
are phosphorylated by glycogen synthase kinase and MAP kinases (compare
results in Refs. 62 and 13). It is possible that one or more of the
DNA-PK-mediated phosphorylation events help to maintain HSF1 in its
active form and prolong its transcriptional activity. This would be
consistent with the higher sustained levels of hsp70 mRNA
transcription that we observed in DNA-PK-positive cells.
A more intriguing possibility is suggested by an analysis of the
sequence defined by the HSF1 If there is competition between DNA-PK and c-Jun N-terminal kinases for
interaction with a common targeting sequence, this could have broad
significance. Hyperthermia causes an increase in ceramide levels, which
in turn activates the stress-activated protein kinase/c-Jun N-terminal
kinase signaling pathway (66-68). This pathway may be the primary
mechanism by which heat induces apoptosis. Interestingly, ionizing
radiation also triggers ceramide release, and
sphingomyelinase-deficient human lymphoblasts and mice are incapable of
undergoing apoptosis even after lethal doses of radiation (69). The
involvement of ceramide-dependent signaling pathways is
thus a common feature of heat- and radiation-induced apoptosis.
Investigation of a possible link between DNA-PK and ceramide-mediated
cell signaling will be a potentially fruitful area for further investigation.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
B, Vav, c-Abl, and certain
transcription factors (8-12), but the physiological significance of
these interactions has not yet been fully explored.
MATERIALS AND METHODS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
-32P]dCTP-labeled
double-stranded oligonucleotide in a total volume of 25 µl. The
sample was incubated for 15 min at 25 °C and analyzed by
nondenaturing gel electrophoresis in 4.5% acrylamide. After electrophoresis, the gel was fixed in 7% acetic acid for 5 min and
dried under vacuum, and complexes containing radiolabeled DNA were
detected by PhosphorImager analysis.
-32P]CTP (10 µCi/ml) was added to begin the labeling of nascent RNA. The mixture
was incubated for 30 min at 30 °C; the reaction was stopped with 540 µl of 0.5 M NaCl, 50 mM MgCl2, 2 mM CaCl2,10 mM Tris-Cl, pH 7.4; and
60 µl of DNase I (1 unit/ml, Promega) were added. A 180-µl aliquot
of 5% SDS, 0.5 M Tris-Cl, pH 7.4, 0.125 M EDTA
and a 20-µl aliquot of 20 mg/ml Proteinase K were added to the
reactions, which were then incubated for 30 min at 42 °C. Samples
were extracted with phenol/chloroform and precipitated with sodium
acetate/ethanol for 20-30 min at
20 °C before centrifugation. The
resulting pellet was resuspended in 225 µl of 20 mM
Hepes, pH 7.5, 5 mM MgCl2, 1 mM
CaCl2, and 25 µl of DNase I (1 unit/ml, Promega), and the
mixture was incubated for 20 min at 37 °C. A 10-µl aliquot of 0.5 M EDTA, 30 µl of 10% SDS, and a 10-µl aliquot of 20 mg/ml Proteinase K were added, and the mixture was further incubated
for 30 min at 30 °C. Samples were extracted with phenol/chloroform and precipitated with ammonium acetate/ethanol. After 2 h at
20 °C, samples were centrifuged, resuspended in 150 µl of TES
buffer (10 mM Tris-HCl, pH 7.4, 10 mM EDTA,
0.2% SDS), and treated with 40 µl of 1 M NaOH for 10 min
at 4 °C. The reactions were stopped by the addition of 75 µl of 1 M Hepes, pH 7.8, and precipitated again with
NH4OAc and ethanol. Samples were centrifuged, resuspended in 150 µl of TES, heated for 5 min at 90 °C, and added directly to
the hybridization solution described below.
-actin cDNA was digested with EcoRV and ScaI to release the actin
structural gene; pT7Zpm70.1, containing the mouse hsp70.1 gene, was
digested with HindIII, StuI, and
BglII; and pT7hsp27, containing the hamster hsp27 cDNA, was digested with ClaI and BamHI. DNA was
fractionated by agarose gel electrophoresis and transferred to a nylon
membrane using 0.4 NaOH (30). Membrane strips were prehybridized for
2 h at 42 °C in a solution containing 5× SSC, 5× Denhardt's
solution, 50% formamide, 1% SDS, and 100 µg/ml denatured salmon
sperm DNA. Membranes were hybridized for 24 h at 42 °C in 10 ml
of the same buffer containing radiolabeled RNA. After hybridization,
membranes were washed for 1 h at 42 °C and 1 h at 65 °C
in a solution containing 2× SSC and 0.1% SDS. Hybridization of
radiolabeled RNA was detected by PhosphorImager analysis.
RESULTS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
Cell lines used in this study
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Fig. 1.
Analysis of reproductive viability
DNA-PK-positive and -negative cells subjected to heat treatment.
Monolayer cloning assays were performed as described under "Materials
and Methods." A, flasks of DNA-PKcs-positive (sc(8)-10)
and DNA-PKcs-negative (scva2) cells were treated for the indicated
times at 43 °C, allowed to recover for 2 h at 37 °C,
trypsinized, and plated. Colonies with 20 or more cells were counted
after 1 week. The surviving fraction was calculated as described under
"Results" section of the text. B, flasks of Ku-positive
(xrs-6cKu80) and Ku-negative (xrs-6cvec) cells were treated for the
indicated times at 43 °C, trypsinized immediately, and plated.
Colonies were counted, and the surviving fraction was calculated as in
A.
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Fig. 2.
Quantitative immunoassay of apoptotic
histone-DNA complexes. A, equal numbers of
DNA-PKcs-positive (sc(8)-10) and DNA-PKcs-negative (scva2) cells were
plated in individual wells of a multiwell plate. Cells were left
unheated (control (ctrl)) or were treated for 60 min at
43 °C and allowed to recover at 37 °C for the indicated times
(heat shock (HS)). Individual wells were harvested, and cell
extracts were assayed by enzyme-linked immunosorbent assay as described
under "Materials and Methods." Points are the average of duplicates
with S.E. indicated. B, same as A but with
Ku80-positive (xrs-6cKu80) and Ku80-negative (xrs-6cvec) cells.
Experiments were performed at least three times for each cell line with
similar results.
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Fig. 3.
Immunofluorescence analysis and quantitation
of apoptotic DNA fragmentation in DNA-PKcs-positive and
DNA-PKcs-negative cells. A, cells were left untreated
(control) or heated at 43 °C for 60 min and allowed to
recover at 37 °C for 4 h (heat shock). Staining was
performed sequentially using fluorescein dUTP/terminal deoxynucleotidyl
transferase labeling and DAPI DNA staining dye. Images in
each row show the same microscope field using different
filters to allow DNA and dUTP to be visualized separately. The
experiment was performed three times, and representative results are
shown. B, quantitation of apoptotic cells was carried out
using microscope fields from two different experiments, including the
field shown in A. Bright dUTP-labeled cells were counted,
and the number was divided by the total number of cells counted in the
DAPI-stained field. Between 365 and 560 total cells were counted for
each condition.
24, which has a deletion from amino acid 280 to 370, binds to DNA-PK but does not stimulate activity (13). HSF1
01, which
has a deletion from amino acid 203 to 224, does not bind to DNA-PK
in vitro (13). These HSF1 derivatives were subcloned into
the pcDNA3 vector, which allows expression under the control of a
cytomegalovirus promoter.
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Fig. 4.
Generation of cell lines stably transfected
with HSF1 derivatives. A, schematic representation of
the transfected constructs with the normal human HSF1 sequence shown as
open boxes and the deleted regions in each
construct shown as solid lines. The known
functional and structural domains of human HSF1 are indicated.
B, expression of the transfected HSF1 constructs in
xrs-6cKu80 cells. Immunofluorescence assays were performed with cells
that were unheated (control) or treated for 1 h at 43 °C and
allowed to recover for 2 h at 37 °C (heat shock). The pattern
of expression in corresponding xrs-6cvec derivatives was essentially
identical (not shown).
01
derivative behaved similarly. Unexpectedly, the HSF1
24 and
HSF1-(1-450) derivatives were localized in the nucleus in both control
and heat-shocked cells. Full-length HSF1 had a pattern of localization
similar to HSF1-(1-450) (not shown). The reason for these differences in localization is unknown but presumably relates to the combination of
overexpression and the presence of the DNA-PK binding site. Similar
experiments were performed to measure expression of HSF1 derivatives in
xrs-6cvec transfectants. Results were comparable with those with
xrs-6cKu80 cells (not shown).
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Fig. 5.
Immunofluorescence analysis and quantitation
of apoptosis in Ku80-positive and -negative cells stably transfected
with HSF1 deletion constructs. A, representative
results using xrs-6cKu80 cells transfected with pcDNA3 or
HSF1-(1-450) as indicated, and xrs-6cvec cells transfected with
pcDNA3 or HSF1-(1-450) as indicated. Cells were unheated (control)
or heat-treated for 1 h at 43 °C and allowed to recover for
4 h at 37 °C (heat shock). Cells were labeled with
dUTP-fluorescein/terminal deoxynucleotidyl transferase and DAPI DNA
staining dye. The same field was analyzed using two different filters.
B, quantitation of apoptosis in xrs-6cKu80 transfectants.
Results are included from cells shown in A, as well as cells
transfected with HSF1 01, HSF1
24, and full-length, wild type
HSF1 (wt). Bright dUTP-labeled cells were counted, and the
number was divided by the total number of cells counted in the
corresponding DAPI-stained fields. In most cases, results from two
different experiments were averaged (except for HSF1 wild type
transfectants, where multiple fields from a single experiment were
averaged). C, the same analysis as in B using
xrs-6cvec cells. In B and C, between 232 and 872 total cells were counted for each condition.
01, which does not interact
with DNA-PK in vivo, did not sensitize xrs-6cKu80 cells to
heat (Fig. 5B). By contrast, expression of two other HSF1
derivatives that bind DNA-PK in vitro, HSF
24 and
full-length HSF1, resulted in an increase in heat sensitivity similar
to that seen with HSF1-(1-450). None of these HSF1 derivatives had any effect in xrs-6cvec cells (Fig. 5C). In addition, none of
the HSF1 derivatives increased apoptosis in unheated control
cells, indicating that their effects are not due to general toxicity.
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Fig. 6.
Induction of endogenous HSF DNA binding
activity and heat shock mRNA synthesis. A,
measurement of HSF DNA binding activity in different cell lines. Equal
numbers of cells were plated in replicate flasks. Cells were left
unheated (control) or were heated for 20 min at 43 °C
(H. shock). Cell extracts were prepared, and equal amounts
of protein were added to binding reactions containing a radiolabeled
double-stranded DNA oligonucleotide with one HSF1 binding site. Where
indicated, reactions also contained a 200-fold excess of
nonradiolabeled oligonucleotide containing a single heat shock element
(200× HSE). Protein-DNA complexes were analyzed by native
gel electrophoresis. The positions of the HSF-DNA complexes are marked
by an arrowhead. B, analysis of nuclear run-on
transcription in DNA-PKcs-positive (sc(8)-10) and DNA-PKcs-negative
(scva2) cells. Equal numbers of cells were plated in replicate flasks.
Cells were left unheated (control) or were subjected to heat treatment,
with times and temperatures as indicated. Nuclei were prepared, and
nascent RNA was radiolabeled by extension in the presence of
[ -32P]CTP. This RNA was purified and hybridized to
membranes to which various plasmid DNA fragments had been blotted as
indicated. The positions of relevant DNA fragments are marked by
arrows. C, analysis of steady state hsp70
mRNA levels. DNA-PKcs-positive (sc(8)-10) and DNA-PKcs-negative
(scva2) cells were left untreated (control) or heated for 30 min at
43 °C and allowed to recover for 2 h at 37 °C (H. shock). Total RNA was purified and quantitated. Equal amounts of
RNA were analyzed by formamide-agarose gel electrophoresis and blotted
to a nitrocellulose membrane as described under "Materials and
Methods." The membrane was hybridized with a probe containing the
coding sequence of human hsp70. The same membrane was stripped and
rehybridized to a probe containing the cDNA coding sequence for
human
-actin.
-32P]CTP, and RNA was hybridized
to a restriction digest of a plasmid containing the mouse hsp70.1 gene
(48). The restriction digest produces three bands, corresponding to the
5'-untranslated region of the mRNA, the coding region, and the
3'-untranslated region, as marked in Fig. 6. We saw a progressive
increase in the hsp70 transcription rate upon heat treatment for 10 or
30 min at 43 °C (Fig. 6B). This initial induction was
similar in both cell lines. The DNA-PKcs-negative cells, however,
showed a somewhat lower rate of hsp70 transcription at the final time
point, after 30 min of heat shock and 2 h of recovery.
-actin. The hsp27 signal was near the limit of
detection in the run-on assay but did not appear to differ in
DNA-PKcs-positive and -negative cells (Fig. 6B).
Transcription of
-actin was shut down in response to heat treatment
in both DNA-PKcs-negative and DNA-PKcs-positive cells (Fig.
6B), consistent with prior work showing that heat treatment
reduces synthesis of non-heat shock mRNAs (reviewed in Refs. 50 and
51).
-actin mRNA was performed as a control (Fig.
6C).
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Fig. 7.
Expression of heat shock proteins in
DNA-PK-positive and -negative cells. A, analysis by
immunofluorescence. Cells were left unheated (control) or heat-treated
for 1 h at 43 °C and allowed to recover for 4 h at
37 °C (heat shock). They were then fixed and stained sequentially
with anti-hsp72/73 antibody, Texas Red-conjugated secondary antibody,
and DAPI DNA staining dye. The same field was analyzed with two
different filters. B, analysis by immunoblotting. Equal
numbers of cells were plated in replicate flasks and were left
untreated (control) or heated for 1 h at 43 °C and allowed to
recover at 37 °C. At the indicated times, individual flasks were
harvested, and cells were lysed in SDS-polyacrylamide gel
electrophoresis sample buffer. Equal amounts of protein were analyzed
by SDS-polyacrylamide gel electrophoresis and immunoblotting using an
anti-hsp72/73 antibody, as described under "Materials and
Methods."
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
01 mutant, which deletes amino acids 204-223 (13). The present
results extend previous findings by suggesting that there may be a
regulatory interaction between HSF1 and DNA-PK in vivo.
Constitutive overexpression of HSF1-(1-450) and certain other
derivatives that are capable of binding DNA-PK suppressed the ability
of DNA-PK to protect against heat-induced apoptosis in vivo.
There was no effect when any of the derivatives were expressed in cells
lacking functional DNA-PK holoenzyme. By contrast, overexpression of
HSF1
01 had no effect. Although we cannot rule out pleiotropic
effects of the HSF1
01 mutation on other HSF1 functions, the lack of
an effect in our experiments is in concordance with the results of
in vitro binding studies.
01 mutant. This sequence,
GVKRKIPLMLNDSGSAHSM, contains a
match (underlined) to a targeting domain, the D domain, which acts as a
binding site for extracellular signal-regulated kinase and c-Jun
N-terminal mitogen-activated protein kinases (64, 65). The D domain,
which is distinct from the actual sites of phosphorylation, is found in
Elk-1 and a number of other transcription factors (64). It is possible
that the binding of DNA-PK to a site overlapping the D domain blocks
the interaction of mitogen-activated protein kinases with HSF1,
preventing inactivation by these kinases. This model could be further
evaluated by competitive binding studies.
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ACKNOWLEDGEMENTS |
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We thank Dr. R. Morimoto for the human hsp70D cDNA. We thank Drs. S. Peterson and D. Chen for providing the cell lines used in this study. We thank Stephen A. Jesch, Wilhelm Woessman, Mark Anderson, Sunghan Yoo, Juren Huang, and Xinbin Chen for helpful suggestions and reagents and Nancy Miller for expert technical assistance. We thank Rhea-Beth Markowitz for comments on the manuscript.
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FOOTNOTES |
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* This work was supported in part by U.S. Public Health Service Grants GM 35866 and CA 62130.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.
Recipient of a postdoctoral fellowship from Spain's Ministerio de
Educación y Ciencia. Present address: CENG DBMS/BRCE, 17 rue des
Martyrs, 38054 Grenoble Cedex 9, France.
§ Georgia Research Alliance Eminent Scholar and recipient of American Cancer Society Faculty Research Award FRA-418. To whom correspondence should be addressed: Institute of Molecular Medicine and Genetics, Medical College of Georgia, 1120 15th St., Augusta, GA 30912. Tel.: 706-721-8756; Fax: 706-721-8752; E-mail: dynan{at}immag.mcg.edu.
2 A. Nueda, F. Hudson, N. F. Mivechi, and W. S. Dynan, unpublished results.
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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; HSF1, heat shock transcription factor 1; hsp, heat shock protein; PBS, phosphate-buffered saline; DAPI, diamidino 2-phenylindole dihydrochloride hydrate; Pipes, 1,4-piperazinediethanesulfonic acid.
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