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INTRODUCTION |
Ataxia telangiectasia
(AT)1 is an autosomal
recessive disorder characterized by immune disorders, acute cancer
predisposition, telangiectasias, sensitivity to ionizing radiation
(IR), and neuronal degeneration (1). The number of individuals who
carry one defective copy of the AT gene has been estimated to be around
0.5-1% of the general population (2). Those AT heterozygotes have
been reported to exhibit elevated cancer risk, particularly for breast cancer and lymphoproliferative disease (3-5). Cultured AT dermal fibroblasts show increased chromosomal instability and acute
sensitivity to IR in comparison to age-matched normal human fibroblasts
(NHFs) (6-10). Cells from individuals with AT exhibit poor p53
induction and severely impaired G1, S, and G2
phase checkpoint functions in response to IR (11-14). The gene mutated
in AT, ATM, has been identified (15), and the gene product,
pATM, has been shown to have IR-inducible protein kinase activity (16,
17). ATM shares homology with the family of
phosphatidylinositol 3'-kinases (15), which include DNA-PK, ATR, MEC1,
TEL1, TOR, and FRAP among others (18). Members of this family of
proteins have been reported to be involved in various aspects of the
detection of DNA damage and control of cell cycle progression (18,
19).
pATM has been suggested to function, at least in part, in the cellular
response to oxidative damage (for review see Ref. 20). Support for this
hypothesis comes from observations that pATM-deficient cells are
unusually sensitive to the toxic effects of hydrogen peroxide, nitric
oxide, and superoxide treatment as determined by colony-forming
efficiency assays. Additionally, they resynthesize glutathione
unusually slowly after depletion with diethyl maleate (21-25).
Furthermore, Barlow and colleagues (26) have shown that ATM-deficient mice have elevated markers of oxidative
stress, particularly in organs such as the cerebellum, which are
consistently affected in individuals with AT. Therefore, we
hypothesized that pATM-deficient fibroblasts would lack normal cell
cycle checkpoint function in response to oxidative stress. To test this
hypothesis, we compared the effect of IR, and reactive oxygen species
produced by treatment with t-butyl hydroperoxide, on normal
and ATM-deficient human fibroblast strains.
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EXPERIMENTAL PROCEDURES |
Cell Cultures and Culture Conditions--
NHF1 is a normal human
fibroblast culture derived from foreskins of apparently healthy
neonates and was used at passages 13-19 (27). GM03349, a normal dermal
fibroblast strain from a 10-year-old male, was obtained from NIGMS
(National Institutes of Health) Human Genetic Mutant Cell Repository
(Camden, NJ) and used at passages 15-19. ATM-deficient
dermal fibroblasts were obtained from the NIGMS Human Genetic Mutant
Cell Repository (strain designations GM02052 and GM03395 (Camden, NJ))
and the NIA Aging Cell Repository (strain AG03058 (Camden, NJ)). The
donor for the cells of strain GM02052 was a 15-year-old Moroccan
female. Cells of this strain contain a mutation at nucleotide 103 that
causes a change in coding from cysteine to thymidine, resulting in a
stop codon at position 35 (28). The cells of strain GM03395 are dermal
fibroblasts cultured from a skin biopsy of a 13-year-old black male,
and the cells of strain AG03058 are dermal fibroblasts cultured from a
skin biopsy from a 14-year-old black female. The exact mutations of ATM in strains GM03395 and AG03058 are not known. However,
no pATM could be detected by immunoblotting in protein extracts from any of these strains, and the donors expressed the typical phenotype of
the AT disease. These cells were used at passages 14-20. Normal human
fibroblasts (NHF1 and GM03349 strains) were grown at 37 °C in a
humidified 5% CO2 atmosphere in minimum Eagle's medium, supplemented with 10% fetal bovine serum (Life Technologies, Inc.) and
2 mM glutamine (Life Technologies, Inc.) (NHF medium).
ATM-deficient fibroblasts (GM02052, GM03395, and AG03058)
were grown under the same conditions in minimum Eagle's medium
supplemented with 20% fetal bovine serum, 2 mM glutamine,
0.4 mM serine, 0.2 mM aspartic acid, and 2 mM pyruvic acid (AT medium) (14). HeLa cells, obtained from
the American Type Culture Collection (Manassas, VA), were cultured as
above in minimum Eagle's medium with 5% fetal bovine serum and 2 mM glutamine. Cells were tested and found to be mycoplasma free.
Logarithmically growing cell populations in 100-mm plastic dishes
(Becton Dickinson Labware, Franklin Lakes, NJ) were exposed to
-rays
at room temperature using a 137Cs source at a rate of 2.6 Gy/min. Mock-treated control cells were subjected to the same movements
both in and out of the incubators as treated cells, for both IR and
t-butyl hydroperoxide treatments. When cells were treated
with t-butyl hydroperoxide, the t-butyl hydroperoxide was added to cell cultures for 15 min. The cultures were
then washed 2× with warm media, and the media were replaced. In
experiments where some cell cultures were treated with
t-butyl hydroperoxide, all plates within the same experiment
were washed as described above. After treatment, the cells were
incubated for the times indicated and harvested.
Cell Colony-forming Efficiency Assay--
Logarithmically
growing fibroblast populations were harvested by typsinization, counted
in a cell counter (Coulter Counter ZM, Coulter Corp., Miami, Fl), and
replated at a density of 103 fibroblasts/100-mm tissue
culture dish. After allowing cells to adhere for 12 h, the
fibroblasts were exposed for 15 min to various concentrations of
t-butyl hydroperoxide, washed, and incubated for 8-12 days
in appropriate media. For assays employing mannitol, fibroblasts were
treated 1 h with 1 mM mannitol, followed by treatment with t-butyl hydroperoxide in the continued presence of 1 mM mannitol, washed, and incubated as described above.
Media were removed and colonies fixed by the addition of water/methanol
(1:1, v/v) containing crystal violet (1 g/liter) and counted using a
dissecting microscope. For each fibroblast strain, a minimum of two
colony-forming assay experiments was performed, with each data point
done in triplicate.
Flow Cytometry for G1 Checkpoint
Function--
G1 checkpoint function was assayed by flow
cytometry using a modification of the cell cycle analysis protocol in
Kastan et al. (1) for simultaneous analysis of DNA synthesis
and cell cycle. Logarithmically growing cells were either mock-treated, exposed to 3.0 Gy
-IR, or exposed to various concentrations of t-butyl hydroperoxide as described above. 4 h following
treatment, BrdUrd (Roche Molecular Biochemicals) was added to the media
to a final concentration of 10 µM, and cells were
incubated for an additional 2 h. Cells were harvested, fixed in
phosphate-buffered saline (PBS)/methanol at a 1:2 (v/v) ratio, and
stored at
20 °C. 5 × 105 cells from each sample
were stained for BrdUrd incorporation in a solution of Tween 20/BSA
plus anti-BrdUrd antibody (Becton Dickinson catalog number 347580) as
recommended by the manufacturer. Fluorescein isothiocyanate-conjugated
anti-mouse IgG antibody (Jackson ImmunoResearch Laboratories, West
Grove, PA) was used as secondary antibody. Cells were stained with PBS
containing 5 µg/ml propidium iodide (Roche Molecular Biochemicals)
and analyzed using a Becton Dickinson FACSort. Twenty thousand cells
were counted for each analysis. Flow cytometric experiments were done
twice with each treatment point done in duplicate.
Protein Analysis and Histone H1 in Vitro Kinase
Assays--
After treatment, cells were harvested and solubilized on
ice in kinase lysis buffer with inhibitors (10 mM sodium
phosphate (pH 7.2), 150 mM NaCl, 1% Nonidet P-40, 1 mM EDTA, 5 mM EGTA, 5 mM
-glycerophosphate, 1 mM dithiothreitol, 120 kallikrein
IU/ml aprotinin, and 10 µg/ml leupeptin). Protein concentration was determined using a detergent-compatible protein assay kit (Bio-Rad) with bovine serum albumin (BSA) as a standard. Although the amount of
protein used in different kinase assay experiments varied, within each
experiment the protein concentrations employed were the same for all
samples. In p34CDC2/cyclin B histone H1 in vitro
kinase activity assays 50-100 µg of protein was used per kinase
reaction, whereas 500-700 µg of protein was used per
p33CDK2/cyclin E histone H1 in vitro kinase
assay. The desired amount of protein from solubilized extracts was
aliquoted into 1.5-ml microcentrifuge tubes, and volumes were
adjusted to 500 µl with kinase lysis buffer. The immunoprecipitations
were done with either 0.5 µl of anti-human cyclin B Powerclonal
antibody (catalog number 05-373, Upstate Biotechnology, Inc., Lake
Placid, NY) or 2.0 µl of anti-human cyclin E Powerclonal antibody
(catalog number 05-371, Upstate Biotechnology, Inc., Lake Placid, NY).
Samples were precleared with protein G-agarose beads (Life
Technologies, Inc.), then incubated with the primary antibody for
2 h, followed by the addition of protein G-agarose beads. Kinase
reactions were carried out in histone H1 kinase buffer (20 mM HEPES (pH 7.3), 80 mM
-glycerophosphate, 20 mM EGTA, 50 mM MgCl2, 5 mM MnCl2, 1 mM dithiothreitol, 60 kallikrein IU/ml aprotinin, 10 µg/ml leupeptin, 10 µM
cyclic AMP-dependent protein kinase-inhibitory peptide),
with 8 µg of histone H1 and 10 µCi of [32
-P]ATP
(3,000 Ci/mmol, Amersham Pharmacia Biotech) for 30 min at 37 °C. The
kinase reactions were stopped by addition of 2× SDS sample buffer (4%
SDS, 150 mM Tris (pH 6.8), 20% glycerol, 1 mM
-mercaptoethanol, 0.02% bromphenol blue), and proteins were resolved by 12% SDS-PAGE. Gels were stained with Coomassie Blue to
verify equal histone protein loading, dried, and subjected to
autoradiography with Hyperfilm MP (Amersham Pharmacia Biotech). The
radiolabeled protein substrates in the dried gels were then quantified
using a Molecular Dynamics PhosphorImager and ImageQuant software.
All kinase assays were performed at least in triplicate.
Quantification of Mitotic Delay Induced by t-Butyl Hydroperoxide
and IR--
After appropriate treatment, fibroblasts were fixed on
100-mm dishes by the gentle addition of cold methanol. After 10 min the
plates were air-dried and stored at 4 °C until staining with 0.2 µg/ml 4',6-diamidino-2-phenylindole (DAPI). DAPI-stained cells then
were examined by fluorescence microscopy. The percentage of mitotic
cells (the mitotic index) was determined from counts of a minimum of
5,000 cells. All mitotic delay treatments were performed in duplicate,
and all experiments were done in duplicate.
Protein Analyses--
Immunoprecipitations of p53 were performed
with whole cell extracts from five 100-mm tissue culture dishes (~800
µg protein/IP) as described above for protein kinase assays, using
2.5 µl of anti-p53 murine monoclonal antibody (catalog number OP03,
Oncogene Research Products, Cambridge, MA) per sample. Eluted proteins were resolved by 12% SDS-PAGE and transferred to 0.2-µm
nitrocellulose membranes. The blots were probed with anti-p53 antibody
(a generous gift from Dr. B. Alex Merrick, NIEHS), which had been
raised in rabbits against immunopurified human r-p53 expressed in a
baculovirus expression system. By using anti-rabbit IgG
peroxidase-conjugated goat antibody (Roche Molecular Biochemicals), p53
protein was visualized by chemiluminescence (Pierce) followed by
exposure to Hyperfilm MP (Amersham Pharmacia Biotech). p53
immunoprecipitations and Western analysis were performed in triplicate.
To examine the effect of t-butyl hydroperoxide on HO-1
protein levels in normal and AT fibroblasts, we treated AT and NHF1 fibroblasts with 1-10 µM t-butyl
hydroperoxide for 4 h. Cells were then harvested and lysed as
described above; 100 µg of whole cell protein extract was loaded per
lane; proteins were resolved by 12% SDS-PAGE and transferred to
nitrocellulose membranes as described above. Nitrocellulose blots were
probed with anti-HO-1 rabbit polyclonal antibody (catalog number
PA3-019, Affinity Bioreagents, Golden, CO) and visualized using
anti-rabbit IgG peroxidase-conjugated antibody (Roche Molecular
Biochemicals) as described above. All HO-1 induction Western blots were
done in triplicate.
Affinity Purification of pATM Antibody,
ATM 7 a.p.--
In order to isolate the highest affinity, highest
specificity
ATM 7 antibodies from the rabbit polyclonal antiserum,
antibody was affinity-purified using the original peptide immunogen.
Peptide corresponding to pATM residues 826-840 (ATM-N826 peptide) was immobilized to generate a peptide column in the following manner. Ten
mg of ATM-N826 peptide were mixed with 1.0 ml of Affi-Gel 15 (Bio-Rad).
The reaction mixture was mixed at room temperature for 2-3 h, and then
the organic solvents were replaced with 10% ethanolamine in water for
1 h at room temperature to terminate the reaction and block any
unreacted groups that remained. The derivatized Affi-Gel was poured
into a column and cleaned by passing 4 M potassium
thiocyanate through the packing. The column was washed with PBS and
stored in PBS with 0.02% sodium azide at 4 °C.
To affinity-purify antipeptide antibody, IgG was isolated from the
whole polyclonal rabbit serum using an Econo-Pac Protein A kit
(Bio-Rad). Twenty five to 30 mg of the protein A-purified IgG was added
to the Affi-Gel peptide column and tumbled overnight at 4 °C. The
antibody-bound peptide beads were washed with 5 column volumes of 1 M potassium thiocyanate, and fractions were eluted with 4 M potassium thiocyanate. BSA (0.1%) was added to peak
fractions prior to dialysis in 1× PBS overnight at 4 °C. The peak
fractions were verified by Western blotting of total protein extracts
from normal and ATM-deficient fibroblasts that had been
resolved by 6% SDS-PAGE (acrylamide/bisacrylamide ratio of 100:1).
Affinity-purified
ATM 7 (
ATM 7 a.p.) antibody was stored in
20% glycerol and 0.02% sodium azide at
20 °C.
pATM in Vitro PHAS-1 Kinase Assay--
The pATM in
vitro PHAS-1 kinase assays were done as described previously (16,
17). Cells were incubated for 90 min following exposure with 6.0 Gy
-IR or a 15-min exposure to 300 µM t-butyl hydroperoxide and then lysed in kinase lysis buffer with 10 mM
-glycerophosphate and 1 mM
NaVO3 added. Cell lysates were clarified by centrifugation;
the protein concentration was determined; the volume was adjusted to
0.9 ml with kinase lysis buffer plus inhibitors, and protein G-agarose
beads were added to pre-clear the lysates as described above. After 30 min, the pre-cleared lysates were removed from the protein G-agarose
beads and added to either 0.1 ml of kinase lysis buffer with inhibitors
alone or containing
ATM 7 a.p. antibody. In order to test
antibody specificity, in some treatments the
ATM 7 a.p.
antibody was incubated for 30 min prior to its addition to the cell
lysates with either 25 µg of the peptide that the antibody was raised
against (ATM-N826) or with 25 µg of an irrelevant peptide sequence of
the same length. All pATM kinase reactions were performed at least in
triplicate using 1.5-3.0 mg protein lysate per immunoprecipitation.
Quantification of Thymine Glycol Formation in ATM-normal and
ATM-deficient Fibroblasts after Treatment with t-Butyl Hydroperoxide
and IR--
To quantify the damage induced in NHFs and
ATM-deficient fibroblasts by t-butyl
hydroperoxide and IR, we measured thymine glycol formation in the DNA
from ATM-normal NHF1 cells and ATM-deficient AG03058 fibroblasts following treatment by each agent. Six plates of
logarithmically growing NHF1 or AG03058 fibroblasts, at ~70% confluence, were treated with 100 or 300 µM
t-butyl hydroperoxide or 6 Gy
-IR, as described above.
Cells were harvested from each plate on ice with 5 ml of cold PBS as
soon as possible following treatment. The cells were pelleted, washed 1 time in cold PBS, re-pelleted in 1.5-ml microcentrifuge tubes,
and quick-frozen in dry ice/ethanol until assayed. Determination of
thymine glycol formation was performed as described previously (29).
Each experimental point was performed in triplicate.
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RESULTS |
AT Fibroblast Strains Are Hypersensitive to the Toxic Effects of
t-Butyl Hydroperoxide--
To examine the relative toxicity of
reactive oxygen species exposure between normal and pATM-deficient
fibroblasts, we treated different fibroblast strains with
t-butyl hydroperoxide. We employed t-butyl
hydroperoxide as a source of oxidative stress as it is poorly
hydrolyzed by catalase (30). We reasoned that this is important since
catalase activity has been reported to be low in
ATM-deficient cells (31, 32) and thus, using a peroxide that
can be hydrolyzed by catalase, would introduce experimental variation
due to differences in cellular catalase activities.
To initiate these studies, two normal and two ATM-deficient
fibroblast strains were treated with increasing levels of IR, and
toxicity was assayed by colony-forming efficiency. As reported previously (7), exposure to increasing amounts of IR inhibited colony
formation in ATM-deficient fibroblast strains more
effectively than in NHFs (Fig.
1A). To compare the effects of
t-butyl hydroperoxide on normal and ATM-deficient
fibroblasts, normal NHF1 and GM03349 cells and ATM-deficient
GM02052 and GM03395 cells were treated with increasing concentrations
of t-butyl hydroperoxide. As shown in Fig. 1B,
the ATM-deficient fibroblasts were more sensitive to the
colony-forming inhibiting effects of t-butyl hydroperoxide than the normal fibroblasts. LC50 (lethal concentration for
50% of population) for both normal fibroblast strains was in the
40-50 µM t-butyl hydroperoxide range, whereas
the LC50 for the ATM-deficient dermal fibroblast
strains was in the 6-8 µM range (Fig. 1B).
The colony-inhibiting effect of t-butyl hydroperoxide in
both normal and ATM-deficient fibroblast strains was
biphasic, with an initial high sensitivity to low concentrations of
t-butyl hydroperoxide (1-10 µM), followed by
less sensitivity at higher concentrations (>10 µM). This
apparent biphasic response is pATM-independent and suggests to us that
higher concentrations of t-butyl hydroperoxide induce a
pATM-independent resistance or adaptive response to the effects of
t-butyl hydroperoxide in both cell types (Fig.
1B, compare 1-10 µM to 10-300
µM t-butyl hydroperoxide).

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Fig. 1.
ATM-deficient cells are hypersensitive to
killing following exposures to IR and t-butyl
hydroperoxide. A, the effect of increasing
concentrations of IR on the colony-forming efficiency of two ATM-normal
and two ATM-deficient fibroblasts. Exponentially growing fibroblasts
were plated at a density of 103 cells/100-mm plate and
allowed to adhere for 12 h. The cells were then treated with
increasing concentrations of -IR and allowed to grow for 8-11 days.
Cell colonies were then stained and counted. Data indicate survival as
a percentage of untreated cells. B, the effect of increasing
concentrations of t-butyl hydroperoxide on the
colony-forming efficiency of two ATM-normal and two ATM-deficient
fibroblast strains. Data indicate survival as a percentage of untreated
cells. C, the effect of 1 mM mannitol on the
colony-forming efficiency of the ATM-normal NHF1 and ATM-deficient
AG03058 fibroblast stains after exposure to 10 and 100 µM
t-butyl hydroperoxide. Data indicate survival as a
percentage of untreated cells.
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Peroxides are thought to exert some of their damaging effects through
the production of reactive oxygen intermediates via events such as the
Fenton reaction (for review see Ref. 33). We pretreated fibroblasts
with mannitol for 1 h prior to t-butyl hydroperoxide
treatment in order to ascertain if the colony-inhibiting effects of
t-butyl hydroperoxide could be reduced by co-treatment with
an antioxidant. Mannitol, which is effective at scavenging hydroxyl
radicals (34), was used as an antioxidant. As shown in Fig.
1C, pretreatment with mannitol partially inhibited the killing effect of t-butyl hydroperoxide on both normal and
ATM-deficient fibroblast strains. For most concentrations
examined, the differences between treatment with and without mannitol
are significant. However, pretreatment with mannitol of the
ATM-deficient cells showed less reduction of killing
following treatment with the highest concentration of
t-butyl hydroperoxide (100 µM) (Fig.
1C). This is likely to be due to the very low number of
ATM-deficient cells that can still form colonies at this
concentration of t-butyl hydroperoxide. Nevertheless, these
data show that the toxic effect of t-butyl hydroperoxide can
be partially reversed by pretreatment with an antioxidant.
Fibroblasts Lacking pATM Function Fail to Exhibit
G1 Checkpoint Delay in Response to t-Butyl Hydroperoxide
Exposure--
G1 checkpoint function, as reflected by
delay of entry into S phase, was assayed following exposure to
oxidative stress. Exposure of NHF1 fibroblasts in logarithmic growth
phase to t-butyl hydroperoxide over a 10-100
µM range resulted in a
concentration-dependent suppression of S phase entry, as
measured by flow cytometry (Table I and Fig. 2). When the
ATM-deficient fibroblast strain AG03058 was subjected to the
same treatment, comparatively little inhibition of S phase entry was
observed over the same concentrations of t-butyl
hydroperoxide (Table I and Fig. 2). As demonstrated previously (11,
35), normal fibroblasts exhibit G1 checkpoint arrest in
response to IR, whereas ATM-deficient fibroblasts did not
(Table I and Fig. 2).
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Table I
Flow cytometric analysis of ATM-normal NHF1 and ATM-deficient AG03058
fibroblasts following treatment with increasing concentrations of
t-butyl hydroperoxide (A) and 3.0 Gy IR (B)
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Fig. 2.
Flow cytometric analysis of G1
checkpoint delay in response to t-butyl hydroperoxide
exposure and IR treatment in ATM-normal NHF1 and
ATM-deficient AG03058 fibroblasts. Exponentially
growing cells were treated as indicated. Four hours after treatment,
BrdUrd (BrdU) was added for the last 2 h of incubation.
Cells were fixed and stained with -BrdUrd-fluorescein isothiocyanate
and propidium iodide. Dot plots show incorporation of BrdUrd
into DNA as an indication of DNA synthesis and propidium iodide
fluorescence as an indication of DNA content. The regions drawn
represent areas from which data were taken for analysis.
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p53 protein stabilization and activation, which is necessary for the
elicitation of a full G1 checkpoint response to cellular damage from IR exposure for more than a transient period (36-38), is
severely attenuated in ATM-deficient cells (35-37). Since
ATM-deficient cells exhibit poor p53 induction in response
to IR and fail to show a G1 checkpoint function in response
to both IR and t-butyl hydroperoxide treatment, we
hypothesized that p53 induction in response to t-butyl
hydroperoxide treatment would similarly be poor in AT fibroblasts
compared with NHFs. When p53 immunoprecipitations were performed,
followed by Western blotting with a second anti-p53 antibody, we found
that p53 was induced in NHF1 cells in response to both
t-butyl hydroperoxide and IR (Fig.
3). p53 induction in response to
t-butyl hydroperoxide was slower than induction in response
to IR, with significant induction following IR exposure by 1 h and
induction by t-butyl hydroperoxide peaking later, ~1-2 h
(Fig. 3). At the time points examined here, neither IR nor
t-butyl hydroperoxide treatment significantly induced p53
protein levels in the ATM-deficient fibroblast strain
GM02052 (Fig. 3).

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Fig. 3.
Western blot of p53 shows induction in
response to t-butyl hydroperoxide or
-IR treatment in normal and
ATM-deficient fibroblasts. Cells were treated as
indicated. p53 was immunoprecipitated with an anti-p53 antibody, and
SDS-PAGE was performed. The resolved proteins were transferred to
nitrocellulose paper and probed with a second anti-p53 antibody.
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p33CDK2/cyclin E-associated kinase activity has been shown
to be required for progression through late G1 and into S
phase (39). To test the effect of t-butyl hydroperoxide on
p33CDK2/cyclin E-associated kinase activity, we treated the
NHF1 fibroblast strain with either 300 µM
t-butyl hydroperoxide or 3.0 Gy IR, followed by incubation
for 1-10 h, and we measured p33CDK2/cyclin E histone H1
in vitro kinase activity. As shown in Fig. 4, both treatments resulted in
p33CDK2/cyclin E histone H1 in vitro kinase
activity suppression. Maximal inhibition occurred at 6 h, followed
by partial recovery at 10 h. The inhibition observed following
treatment of NHF1 cells with 300 µM t-butyl
hydroperoxide was less than that with exposure to 3.0 Gy IR and
occurred with delayed kinetics compared with IR treatment (compare Fig.
4, A and B). When NHF1 cells were treated with
t-butyl hydroperoxide over a 10 µM to 1 mM range and incubated for 6 h,
p33CDK2/cyclin E histone H1 in vitro kinase
activity was found to fall in a concentration-dependent
manner, with kinase activity reduced ~70% at concentrations of 100 µM t-butyl hydroperoxide and above (data not
shown). When the ATM-deficient dermal fibroblast strain GM02052 was treated with 300 µM t-butyl
hydroperoxide or 3.0 Gy, followed by incubation for 1-10 h, the
ATM-deficient fibroblasts failed to exhibit significant
inhibition of kinase activity by either agent (Fig. 4, A and
B).

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Fig. 4.
Time course showing the effect of
t-butyl hydroperoxide and IR treatment on
p33CDK2/cyclin E kinase activity in a normal and
ATM-deficient fibroblast strain. Cells in log
phase were exposed for 15 min to 300 µM
t-butyl hydroperoxide (A) or exposed to 3.0 Gy
-IR (B). The cells were then cultured for the indicated
times, lysed, and immunoprecipitated with anti-cyclin E antibody to
obtain p33CDK2/cyclin E protein complexes following the
addition of protein G-agarose beads. p33CDK2/cyclin E
kinase activity was measured by in vitro kinase assay using
exogenous histone H1 protein as the substrate in the presence of
[ -32P]ATP. SDS-PAGE was performed to resolve proteins;
the gel was dried, and kinase activity was quantified by
phosphorimaging.
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t-Butyl Hydroperoxide Exposure Induces a G2 Checkpoint
Response in Normal Fibroblasts That Is Defective in Fibroblasts Lacking
pATM Function--
Exposure of logarithmically growing cells from the
normal fibroblast strain NHF1 to 1.5 Gy IR resulted in a rapid delay of entry into mitosis 2 h post-treatment, with a reduction in the mitotic index to only 3% (±10%) that of the mock-treated cells. ATM-deficient fibroblast cells (GM02052) showed no
significant reduction in the mitotic index relative to mock-treated
controls (94 ± 2%), in agreement with previous findings (40).
Similarly, exposure of NHF1 cells to 10-300 µM
t-butyl hydroperoxide generated a strong G2
checkpoint response (Fig. 5). Under the
same conditions over the same concentration range of t-butyl
hydroperoxide treatment, no significant inhibition of entry into
mitosis was observed with the ATM-deficient fibroblast
strain cells (GM02052) 2 h post-treatment (Fig. 5).

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Fig. 5.
Analysis of the delay of mitotic entry
following exposure to different concentrations of
t-butyl hydroperoxide in the
ATM-normal NHF1 and ATM-deficient
GM02052 fibroblasts. Cells were treated as indicated, fixed by the
addition of cold methanol, and stained with DAPI. The percentage of
mitotic cells was counted by fluorescence microscopy. The results are
expressed as the relative mitotic index, which is the mitotic index of
the treated population expressed as a percentage of the mitotic index
of the mock-treated population.
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To explore further the G2 checkpoint response to
t-butyl hydroperoxide treatment, we performed
p34CDC2/cyclin B histone H1 in vitro kinase
activity assays on protein extracts from two normal and two
ATM-deficient fibroblast strains. As shown in Fig.
6A, p34CDC2/cyclin
B histone H1 in vitro kinase activity was suppressed in a
concentration-dependent manner 2 h post-treatment with
1-300 µM t-butyl hydroperoxide in both normal
human fibroblast strains, NHF1 and GM03349. Similarly, exposure to 1.5 Gy IR inhibited p34CDC2/cyclin B histone H1 in
vitro kinase activity from normal human fibroblasts, as we have
previously reported (40, 41) (Fig. 6B). Neither treatment
with t-butyl hydroperoxide at any concentration nor exposure
to 1.5 Gy IR caused the two ATM-deficient fibroblast strains, GM02052 and GM03395, to display a significant suppression of
p34CDC2/cyclin B histone H1 in vitro kinase
activity (Fig. 6, A and B). A representative
p34CDC2/cyclin B histone H1 in vitro kinase
assay for NHF1 and GM02052 fibroblasts is shown in Fig.
6C.

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Fig. 6.
Concentration-response curve showing the
effect of t-butyl hydroperoxide and
-IR treatment on p34CDC2/cyclin B1
kinase activity in two ATM-normal and two
ATM-deficient fibroblast strains. Cells were
harvested for analysis 2 h following treatment with either
increasing concentrations of t-butyl hydroperoxide
(A) or 1.5 Gy -radiation (B). Cyclin
B1-associated kinase activity was analyzed by immunoprecipitation of
protein with anti-cyclin B1 antibody, followed by in vitro
kinase analysis using histone H1 as an exogenous substrate.
C shows a representative p34CDC2/cyclin B1
kinase assay with pATM-normal NHFs and pATM-deficient GM02052
fibroblasts. Proteins were resolved by SDS-PAGE, and incorporation of
-32P into histone H1 protein was quantified by
PhosphorImager analysis.
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To investigate the possibility that the ATM-deficient
fibroblast strains were capable of exhibiting a G2
checkpoint function but with delayed kinetics in response to exposures
of t-butyl hydroperoxide, a time course analysis of
p34CDC2/cyclin B histone H1 in vitro kinase
activity following treatment was performed. The results indicate that
treatment with 300 µM t-butyl hydroperoxide
and exposure to 1.5 Gy IR were both effective at suppressing
p34CDC2/cyclin B histone H1 kinase activity in NHF1 cells
at 1 h post-treatment, with maximal suppression of kinase activity
occurring at 2 h. Activity recovered to roughly untreated levels
by 6 h (Figs. 7, A and
B). These kinetics closely resemble those reported
previously for the suppression of mitotic entry by 1.5 Gy IR (40).
Neither ATM-deficient fibroblast strain (GM02052 and
GM03395) showed significant suppression of p34CDC2/cyclin B
histone H1 in vitro kinase activity in response to either treatment with 300 µM t-butyl hydroperoxide or
exposure to 1.5 Gy IR at any time points examined (Fig. 7, A
and B). Mannitol pretreatment reduced the
p34CDC2/cyclin B histone H1 in vitro kinase
activity inhibition initiated by 100 µM
t-butyl hydroperoxide treatment (data not shown). Thus an
antioxidant may partially reverse the suppressive effects of t-butyl hydroperoxide on the G2 checkpoint
function as indicated by the inhibition of p34CDC2/cyclin B
kinase activity.

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Fig. 7.
Time course showing the effect of
t-butyl hydroperoxide and -IR
treatment on p34CDC2/cyclin B1 kinase activity in two
ATM-normal and two ATM-deficient
fibroblast strains. Cells were exposed to either 300 µM t-butyl hydroperoxide (A) or 1.5 Gy -radiation (B) and harvested at the indicated times.
Cyclin B1-associated kinase activity was analyzed using histone H1 as
an exogenous substrate.
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t-Butyl Hydroperoxide Treatment Activates pATM-associated Kinase
Activity--
IR treatment of melanoma and lymphoblast cell lines was
found to increase pATM-associated kinase activity toward the PHAS-1 and
p53 proteins in in vitro kinase activity assays (16, 17). IR-inducible kinase activity was found in pATM normal cell lines but
not in cells lines derived from AT patients. In these studies, pATM
kinase activity was reported to be induced by roughly 2-fold following
treatment with IR (16, 17). Based on the data above, we hypothesized
that oxidative damage generated by treatment with t-butyl
hydroperoxide should induce pATM-associated kinase activity.
A rabbit polyclonal antiserum to a peptide corresponding to pATM
residues 826-840 was raised and affinity-purified to the cognate
peptide. Based on Western blot analysis of whole cell and/or nuclear
protein extracts from NHF1 and HeLa cells, it was determined that the
anti-ATM 7 affinity-purified antibody (
ATM 7 a.p.) recognizes a
protein of ~350 kDa, the predicted size of pATM. This band was not
detected in dermal fibroblasts derived from individuals with AT (Fig.
8).

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Fig. 8.
pATM Western analysis. HeLa cells, NHF1
cells, and two dermal fibroblast strains from individuals with AT were
analyzed by immunoblot analysis for pATM. Whole cell extracts were
prepared and subjected to SDS-PAGE followed by transblotting onto
nitrocellulose. The blots were then probed for pATM using ATM 7 a.p., a rabbit polyclonal antibody that was peptide
affinity-purified.
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NHF1 cells and the ATM-deficient dermal fibroblast strain
AG03058 treated with either 6.0 Gy IR or 15 min with 300 µM t-butyl hydroperoxide were examined for
pATM kinase activity 90 min following treatment. As shown in Fig.
9A, low levels of
pATM-associated in vitro kinase activity were found to be
associated with immunoprecipitated protein complexes from extracts from
untreated NHFs using the
ATM 7 a.p. antibody. This activity was
roughly 50% higher than the background level of kinase activity
associated with protein G-agarose bead mock immunoprecipitations (using
no antibody) with extracts from IR-treated NHFs. When protein extracts
from NHF1 cells treated with either IR or t-butyl
hydroperoxide were assayed for pATM-associated in vitro
kinase activity toward PHAS-1 protein, the level of activity in the
immunocomplexes was significantly increased. t-Butyl
hydroperoxide treatment induced pATM-associated kinase activity an
average of 2.2-fold over the level found associated with pATM
immunocomplexes from untreated NHF1 cells in 3 independent experiments.
IR treatment increased pATM-associated kinase activity an average of
2.1-fold (average of 6 independent experiments) (Fig. 9A). A
representative pATM kinase assay is shown in Fig. 9B. To
examine the pATM-dependent specificity of these in
vitro kinase assays,
ATM 7 a.p. antibody was preincubated
with either the peptide that it was raised against (ATM-N826) or an
irrelevant peptide of the same length (data not shown). As shown in
Fig. 9A, the ATM-N826 peptide largely blocked IR-induced
pATM-associated in vitro kinase activity to PHAS-1. Finally,
pretreatment with 1 mM mannitol significantly lowered
t-butyl hydroperoxide-induced pATM-associated kinase
activity toward the PHAS-1 protein (data not shown), indicating that an
antioxidant could reverse the pATM-activating effects of
t-butyl hydroperoxide in this assay.

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Fig. 9.
pATM in vitro kinase
assay. A, NHFs (ATM-normal) and the
ATM-deficient dermal fibroblasts AG03058 were treated with
either 300 µM t-butyl hydroperoxide or 6.0 Gy
-IR, as indicated, incubated for 90 min, then harvested, and lysed,
and protein extracts were subjected to immunoprecipitation with ATM
7 a.p. antibody. An in vitro kinase analysis was
performed using PHAS-1 protein as an exogenous substrate in the
presence of [ -32P]ATP. Error bars in this
figure represent standard deviation in three successive pATM in
vitro kinase assays. The incorporation of -32P into
PHAS-1 protein was quantified by SDS-PAGE followed by PhosphorImager
analysis. B, a representative pATM in vitro
kinase assay used to generate figure A. The relative values
of each data point are as follows: 1st lane, 1.00 ± 0;
2nd lane, 1.55 ± 0.19; 3rd
lane, 3.24 ± 1.54; 4th lane,
3.35 ± 0.90; 5th lane, 1.46 ± 0.75;
6th lane, 1.00 ± 0; 7th lane,
1.08 ± 0.33; and 8th lane, 1.02 ± 0.27, where assays with extracts from -irradiated cells plus protein
G-agarose beads without any antibody were set at 1.00 in each
experiment.
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Further confirmation that the in vitro kinase activity
toward PHAS-1 was associated with pATM was found in that in
vitro kinase activity assayed from protein extracts from
fibroblasts lacking pATM showed no significant difference whether
anti-pATM antibody was present in the assays or not. Furthermore, with
extracts from IR- or t-butyl hydroperoxide-treated
ATM-deficient fibroblasts, the addition of ATM-N826 peptide
to the assay did not suppress the background level of in
vitro kinase activity (Fig. 9B).
Heme Oxygenase-1 Induction Is Normal in AT Fibroblasts--
Heme
oxygenase-1 (HO-1) is induced in human skin fibroblasts by peroxides
(42). To determine whether AT fibroblasts were deficient in this
response to oxidative stress, we treated four fibroblast strains (1 normal and 3 from individuals with AT) with 0, 1, 3, 10, 30, and 100 µM t-butyl hydroperoxide for 4 h and performed Western blot analysis for HO-1 protein. As shown in Fig.
10, t-butyl hydroperoxide
treatment resulted in an induction of HO-1 protein at 10 µM concentrations and above in all four fibroblast
strains. There was no significant difference in the response of any of
the fibroblasts to HO-1 induction. Western blot analyses demonstrated
that t-butyl hydroperoxide maximally induced HO-1 protein at
4-5 h in each cell type (data not shown). Pretreatment of NHF1
fibroblasts with mannitol for 1 h followed by treatment with 10 µM t-butyl hydroperoxide for 4 h in the
continued presence of mannitol resulted in a significant inhibition of
HO-1 protein induction, demonstrating that an antioxidant could
partially reverse the effects of t-butyl hydroperoxide (data
not shown).

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Fig. 10.
Western blot analysis of heme oxygenase
induction in response to t-butyl hydroperoxide
treatment. One normal and three ATM-deficient
fibroblast strains were treated as indicated; whole cell lysates were
prepared, and protein extracts were subjected to SDS-PAGE. The resolved
proteins were then transblotted onto nitrocellulose and probed using an
anti-heme oxygenase antibody.
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Thymine Glycol Formation Induced by t-Butyl Hydroperoxide Treatment
Is the Same in pATM-normal and pATM-deficient Fibroblasts--
NHF1
cells and ATM-deficient fibroblasts (AG03058) were treated
with t-butyl hydroperoxide or IR and examined for thymine glycol formation. As shown in Table II,
treatment of either fibroblast type with 100 or 300 µM
t-butyl hydroperoxide (A) or 6.0 Gy
-IR (B) resulted in
essentially equal thymine glycol formation between the fibroblast
types. Thus, the initial damage produced by t-butyl hydroperoxide and IR treatment in pATM normal and deficient fibroblasts is not significantly different, as measured by thymine glycol formation.
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Table II
Thymine glycol formation in ATM-normal NHF1 and ATM-deficient AG03058
fibroblasts following treatment with 100 and 300 µM
t-butyl hydroperoxide and 6.0 Gy -IR
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DISCUSSION |
We have examined the role of pATM in cellular responses to
oxidative damage generated by t-butyl hydroperoxide
treatment of normal human fibroblasts and ATM-deficient
dermal fibroblasts. We undertook this study for several reasons as
follows. 1) ATM-deficient cells have been reported to be
unusually sensitive to oxidants such as nitric oxide, superoxide, and
hydrogen peroxide in colony-forming efficiency assays compared with
normal cells (21, 23-25). 2) ATM-deficient cells
re-synthesize glutathione unusually slowly after depletion with diethyl
maleate (22). 3) Cellular damage by IR exposure has been suggested to
involve damage due to reactive oxygen species (43). 4) The
RAD9 gene product, which has similar functions to
pATM, has been found to be necessary for checkpoint arrest in response
to peroxide exposure (44). 5) BRCA1, which has been demonstrated to
play a role in protecting cells from damage by hydrogen peroxide (45),
has recently been found to be phosphorylated by activated pATM (46). 6)
ATM-deficient mice have been found to have elevated markers
of oxidative damage, such as nitrotyrosine and hemeoxygenase activity,
in organs known to be affected by the AT phenotype. Interestingly,
cerebellar HO levels were found to be 600% greater in
ATM-deficient mice than in ATM-normal mice (26).
This last observation is particularly interesting as cerebellar
pathologies are a hallmark of AT (47-49). Finally, pATM has been
suggested to function, in part, as a sensor of oxidative stress (20).
We report that compared with pATM normal fibroblasts,
ATM-deficient fibroblasts exhibit the following
characteristics after oxidative damage: 1) greater sensitivity to the
toxic effects of t-butyl hydroperoxide treatment in
colony-forming efficiency assays; 2) failure to delay late
G1 phase progression, as measured by flow cytometry; 3)
failure to exhibit G1 checkpoint function, as measured by
inhibition of p33CDK2/cyclin E-associated in
vitro kinase activity; 4) failure to induce p53; 5) failure to
exhibit inhibition of p34CDC2/cyclin B histone H1 in
vitro kinase activity; and 6) failure to delay entry into mitosis.
Also t-butyl hydroperoxide stimulates pATM-associated kinase
activity. Thus, in response to oxidative damage,
ATM-deficient cells appear to lack many critical cell cycle
checkpoint responses, in a similar manner to that observed with
IR-induced damage (for review see Ref. 33).
In contrast to these findings, HO-1 protein induction in response to
t-butyl hydroperoxide appeared normal in
ATM-deficient fibroblasts, indicating that some cellular
responses to oxidative damage are regulated normally in
ATM-deficient cells. Similarly, both
ATM-deficient and -normal cells exhibited a biphasic
response in colony-forming efficiency assays, with an initial
sensitivity to low concentrations of t-butyl hydroperoxide
(1-10 µM), followed by lesser sensitivity at higher
concentrations (>10 µM). Whereas ATM-deficient fibroblasts were clearly more sensitive to the
colony-forming inhibitory effects of t-butyl hydroperoxide,
the appearance of the biphasic response in both normal and AT cells
suggests that higher concentrations of t-butyl hydroperoxide
(>10 µM) induce a resistance to the effects of
t-butyl hydroperoxide that is both pATM-independent and
similar to that seen in normal cells. The biphasic colony-inhibitory
response we found here is similar to one reported with mouse embryonic
stem cells exposed to hydrogen peroxide (45), suggesting that this
biphasic response may be a common adaptive event following peroxide exposure.
One possible interpretation of our findings could be that
ATM-deficient cells may respond normally to oxidative damage
generated by t-butyl hydroperoxide treatment but not exhibit
normal checkpoint responses or p53 induction due to their receiving
less damage than normal fibroblasts after exposure to the same
concentration of t-butyl hydroperoxide. To address this
issue we quantified DNA damage induced by IR and t-butyl
hydroperoxide exposure by quantifying thymine glycol formation in
normal and ATM-deficient fibroblasts. We found that thymine
glycol formation was not significantly different between the fibroblast
types directly following exposure to either agent. Based on these data,
we conclude that the initial damage received by normal and
ATM-deficient fibroblasts following IR and
t-butyl hydroperoxide exposure is approximately the same and
that, in fact, it is the absence of pATM function that accounts for the
differences seen between the cells types.
A major hallmark of cells from individuals with AT is the ablation of
the G1, S, and G2 checkpoint functions in
response to IR, as well as other IR response-related events, such as
p53 and p21 induction (11, 12, 36, 37, 50). Here we have shown that
after exposure to t-butyl hydroperoxide,
ATM-deficient fibroblasts lack G1 and
G2 checkpoint responses, lack p53 induction, and show enhanced toxicity, reminiscent of the cellular responses seen with
ATM-deficient fibroblasts exposed to IR. Thus our data
support the hypothesis that pATM plays a role in resistance to
oxidative stress (20).
Cell cycle checkpoints are thought to function, in part, to allow cells
time to repair damage, particularly damage to DNA, before re-entering
the cell cycle and completing subsequent cellular events such as DNA
replication or mitosis. Ablation of cellular checkpoint functions, such
as occurs in ATM-deficient cells or in cells treated with
methylxanthines, can result in greater lethality following exposure to
toxic agents (for review see Ref. 33). One question we have not
addressed is whether or not pATM responds to oxidative damage to DNA or
responds directly to a change in intracellular redox state independent
of DNA damage. Although our data do not address this question
specifically, we do demonstrate that ATM-deficient
fibroblasts fail to exhibit normal G1 and G2 checkpoint responses following exposure to oxidative stress. Thus the
inability of ATM-deficient cells to exhibit G1
and G2 checkpoint responses following oxidative damage is
likely to be one mechanism that predisposes these cells to enhanced
toxicity in response to exposure to oxidative stress. Our results
suggest that there is an inability to respond to oxidative stress in
cells from individuals with AT, a consequence of which may be relevant
to the mechanisms of Purkinje cell death and other pathological changes
observed in AT patients.