From the Department of Pharmacology, Program in Molecular and Cellular Oncology, The George Washington University Medical Center, Washington, D. C. 20037
Received for publication, October 15, 2002, and in revised form, March 7, 2003
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ABSTRACT |
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The ataxia telangiectasia
mutated (ATM) protein plays a central role in early stages of DNA
double strand break (DSB) detection and controls cellular responses to
this damage. Although hypersensitive to ionizing radiation-induced
clonogenic lethality, ataxia telangiectasia cells are paradoxically
deficient in their ability to undergo ionizing radiation-induced
apoptosis. This contradiction illustrates the complexity of the central
role of ATM in DNA damage response and the need for further
understanding. Certain hexavalent chromium (Cr(VI)) compounds are
implicated as occupational respiratory carcinogens at doses that are
both genotoxic and cytotoxic. Cr(VI) induces a broad spectrum of DNA
damage, but Cr(VI)-induced DSBs have not been reported. Here, we
examined the role of ATM in the cellular response to Cr(VI) and found
that Cr(VI) activates ATM. We also show that physiological targets of
ATM, p53 Ser-15 and Chk2 Thr-68, were phosphorylated by Cr(VI) exposure
in an ATM-dependent fashion. We found that ATM Ataxia telangiectasia
(AT)1 is an autosomal
recessive disorder characterized by progressive cerebellar
degeneration, immunodeficiency, hypersensitivity to ionizing radiation
(IR), and cancer predisposition (1). The gene responsible for this
pleiotropic phenotype, ATM, is inactivated in the majority of AT
patients. The clinical symptoms of AT are attributed to the cellular
defects observed in cell lines derived from AT patients, which include
chromosomal instability, telomere end-to-end fusion, as well as defects
in cell cycle checkpoints (2, 3), and delayed or attenuated activation
of damage response pathways after exposure to IR (3). The product of
the ATM gene is a 370-kDa serine/threonine kinase (4). The
COOH-terminal domain of the ATM protein, which harbors the catalytic
site of ATM, shows high similarity to the catalytic domain of
phosphatidylinositol 3-kinases. This domain assigns ATM to a growing
family of large proteins that contain the phosphatidylinositol
3-kinase-related domain (phosphatidylinositol 3-kinase-related kinases)
(4). These proteins, which include ATM, ataxia telangiectasia mutation and Rad3-related kinase (ATR), and DNA-dependent protein kinase (DNA-PK), are involved, to different extents, in cell cycle
progression, cellular responses to DNA damage, and maintenance of
genomic integrity (5). The ATM protein has been shown to function
specifically in multiple biochemical pathways linking the recognition
and repair of DNA double strand breaks (DSBs) to downstream cellular
processes, such as activation of cell cycle checkpoints, DNA repair,
and apoptosis (6) by phosphorylating numerous substrates including p53
(7), Brca1 (8), Chk2 (9), c-Abl (10), and replication protein A
(11).
AT cells have a higher level of spontaneous apoptosis but appear to be
deficient in their ability to undergo apoptosis in response to IR (12,
13). The deficiency in apoptosis is not caused by a defect in the
apoptotic machinery because these cells undergo apoptosis in response
to FAS ligand (12, 13). Interestingly, AT cells are hypersensitive to
IR-induced clonogenic lethality (14). The contradiction between the
hypersensitivity to clonogenic lethality and deficiency in apoptotic
cell death in response to IR illustrates the complexity of the central
role of ATM in the DNA damage response and the need for further
understanding. ATM kinase has been shown to be specifically activated
in response to DNA DSBs but not to UVB/UVC or base-damaging agents (7). However, recent reports have demonstrated the activation of ATM by
t-butylhydroperoxide (15), CdCl2 (16), UVA
(17), heat shock (18), insulin (19), and MNNG (20). Therefore, the function of ATM in the cellular response to DNA damage other than DSBs
also needs further investigation. Here, we studied the role of ATM in
the cellular response to hexavalent chromium (Cr(VI)).
Cr(VI) has been shown to be a complex genotoxin and induce a broad
spectrum of DNA damage. The spectrum of structural DNA damage includes
DNA-DNA cross-links (21-23), DNA monoadducts (24, 25), and DNA-protein
cross-links (25). Cr(VI)-induced DNA single strand breaks appear when
DNA is isolated under alkaline conditions (26). Despite the wide range
of DNA damage induced by Cr(VI), no evidence has linked Cr(VI) to
direct DSB formation; however, Cr(VI) is a potent inducer of
recombination (27). We have shown that Cr(VI) exposure results in
activation of p53 (28) and that Cr(VI)-induced apoptosis is
p53-dependent (29). However, the upstream signal
transducers of p53 activation remain unclear. Furthermore, in a recent
study, we showed that telomerase-immortalized human cell populations
exposed to Cr(VI) exhibit a different spectrum of responses, which
include clonogenic survival, terminal growth arrest or apoptosis,
depending on the extent of DNA damage. The regaining of replicative
potential after genotoxic exposure was attributable to either escape
from, or resistance to, terminal growth arrest or apoptosis (30).
The objectives of this study were to characterize the role of ATM in
the cellular response to Cr(VI) as a model complex genotoxin. We showed
that ATM is activated after Cr(VI) treatment. ATM Cell Culture--
Normal dermal fibroblasts GM03440, AT
heterozygous fibroblasts GM03397, AT fibroblasts GM03395 with
homozygous ATM gene deletion (the donor of this cell line is also the
son of the donor of GM03397), and GM02052 with a homozygous point
mutation for the 103C-T transition in exon 5 of the ATM gene (Coriell
Institute, Camden, NJ) were maintained in minimum essential medium with
Earle's salts (EMEM). The EMEM contained a 2× concentration of
essential and nonessential amino acids, vitamins, and 20% fetal bovine
serum (Hyclone Laboratories, Inc., Logan, UT). Cells were incubated in
a 95% air and 5% CO2 humidified atmosphere at 37 °C,
and the medium was replaced every 48 h.
Treatment of Cells with Chromium--
Sodium chromate
(Na2CrO4.4H2O) (J. T. Baker Chemical Company) was dissolved in deionized H2O
and sterilized by passage through a 0.2-µm filter before use. Cells
were treated with a final concentration of 0, 0.1, 0.5, 1, 3, or 6 µM sodium chromate for the indicated times in complete
medium. The cells were either collected for analysis after this
treatment or rinsed with phosphate-buffered saline (PBS) and incubated
in complete medium for an additional 24 h before analysis.
Western Blot--
Equal numbers of experimental cells (1 × 106-12 × 106 cells) were cultured for
24 h in 100-mm or 150-mm dishes. At the indicated times after
Cr(VI) treatment, the cells were harvested and lysed by sonication in
TGN buffer (50 mM Tris (pH 7.5), 50 mM
glycerophosphate, 150 mM NaCl, 10% glycerol, 1% Tween 20, 1 mM NaF, 1 mM NaVO4, 1 mM phenylmethylsulfonyl fluoride, 2 µg/ml pepstatin A, 5 µg/ml leupeptin, 10 µg/ml aprotinin, and 1 mM
dithiothreitol) (7). For detection of cleaved caspase-3, cells were
lysed by freezing and thawing three times in CHAPS cell lysis buffer
(Cell Signaling Technology, Beverly, MA). The cell lysates were then
clarified by microcentrifugation, and the supernatant fractions were
saved. The samples were resolved by 5% (for ATM) or 10% (for
phospho-p53 Ser-15, total Chk2, and phospho-Chk2 Thr-68) or 15% (for
cleaved caspase-3) SDS-PAGE and transferred to polyvinylidene
difluoride membranes (Bio-Rad). The membranes were probed with
polyclonal ATM antibody, polyclonal Chk2 antibody (Calbiochem),
polyclonal p53-phosphorylated Ser-15 antibody, polyclonal
Chk2-phosphorylated Thr-68 antibody, or polyclonal cleaved caspase-3
antibody (Cell Signaling Technology) overnight. The membranes were then
probed with a horseradish peroxidase-linked secondary antibody
(Amersham Biosciences) for 1 h. The secondary antibody was
visualized by ECL Western blotting detection reagents (PerkinElmer Life
Sciences). Equal protein loading was confirmed by immunoblotting with
an antibody to constitutively expressed G3PDH (Trevigen, Gaithersburg, MD).
ATM in Vitro Kinase Assay--
12 × 106 Cells
were treated with and without 6 µM sodium chromate for
3 h. Cells were then lysed by sonication in TGN buffer. After
centrifugation at 13,000 × g, 1.5 mg of extracted
proteins was incubated with 30 µl of mouse IgG-agarose (Sigma) for
1 h. ATM protein was then immunoprecipitated with anti-ATM
polyclonal antibody and protein A-agarose (Calbiochem). The
immunocomplexes were recovered by centrifugation at 13,000 × g for 20 s and washed twice with TGN buffer, twice with
TGN buffer plus 0.5 M LiCl, and twice with kinase buffer
(150 mM NaCl, 10 mM MnCl2, 1 mM dithiothreitol, 1 mM NaF, 1 mM
NaVO4, 5 µM ATP). Kinase reactions were
initiated by resuspending washed beads in 30 µl of kinase buffer
containing 10 µCi of [ Phosphatidylserine (PS) Translocation Assay--
This assay
measures PS translocation from the inner (cytoplasmic) leaflet of the
plasma membrane to the outer (extracellular) leaflet in the early
stages of apoptosis. PS on the outer leaflet is available for binding
to fluorescently labeled annexin V (31). This assay was conducted as
described previously (28). Briefly, cells were seeded at
105 cells/60-mm2 dish and incubated for 24 h prior to sodium chromate exposure. After the sodium chromate
treatment, cells were gently harvested by trypsinization and combined
with nonadherent cells from the culture medium. The cells were
centrifuged at 600 × g for 5 min. Cell pellets were
washed once with PBS and resuspended in 100 µl of binding buffer (10 mM HEPES (pH 7.4), 150 mM NaCl, 5 mM KCl, 1 mM MgCl2, 1.8 mM CaCl2) containing 2 µl of annexin
V-FLUOSTM (Roche Applied Science). Samples were incubated in the dark
at room temperature for 15 min. 30 µl was loaded on a microscope slide, and the percentage of annexin V-FLUOS-stained cells was determined using an Olympus AX70 microscope (Olympus, Lake Success, NY)
with a fluorescent filter set suitable for FLUOS analysis (excitation
460-490, emission 515).
Clonogenicity Analysis--
This assay was conducted as
described previously (30). Briefly, cells were seeded at
105 cells/T25 flask and treated with 0, 0.1, 0.5, and 1 µM sodium chromate. After 24 h, cells were collected
by trypsinization, counted, and reseeded at 2 × 103/60-mm2 dish in triplicate. The cells were
incubated for 12 days, then rinsed with PBS and incubated with crystal
violet stain (80% methanol, 2% formaldehyde, 2.5 g/liter crystal
violet) for 15 min at room temperature. The plates were rinsed
thoroughly with distilled H2O and allowed to dry. Colonies
with 20 or greater cells were counted, the triplicates were averaged,
and clonogenicity was determined as percent of control.
Cell Growth Analysis--
Cells were seeded at 105
cells/100-mm dish and treated with 0, 0.1, 0.5, and 1 µM
sodium chromate. A group of nine replicate dishes were seeded for each
dose tested, and all of the replicates within the group received the
same treatment. One replicate was taken at day 1-6, 8, 12, and 16 after Cr(VI) treatment and counted to determine total cell number at
each dose and time. Cells were gently harvested with trypsin,
centrifuged at 600 × g for 5 min, and the cell pellets
were resuspended in 1 ml of PBS. The total cell number was determined
using a hemacytometer (Hausser Scientific, Horsham, PA). Data were
normalized to the same day nontreated control. PS translocation assay
was conducted at days 1, 8, 12, and 16 to determine the possible cell
death induced by 1 µM Cr(VI) treatment.
Cell Cycle Analysis Using Bromodeoxyuridine (BrdUrd) and
Propidium Iodide (PI) Double Staining--
Cells were seeded at 4 × 105 cells/150-mm dishes and incubated with or without 1 µM Cr(VI) for 24 h and then washed and maintained in
complete medium. At the indicated time after Cr(VI) treatment, BrdUrd
(Molecular Probes, Eugene, OR) was added to the medium to a final
concentration of 10 µM, and cells were incubated for an
additional 1 h. Cells were then harvested and fixed in 70% ethanol. After fixation, cells were resuspended in 2 N HCl
for 20 min and neutralized in 0.1 M sodium borate. Cells
were then incubated with a 1:40 dilution of anti-BrdUrd antibody for 30 min. Alexa 488-conjugated anti-mouse IgG antibody was used as a
secondary antibody (Molecular Probes). Cells were stained with 5 µg/ml PI (Sigma) in PBS and analyzed using a FACSort flow cytometer (BD Biosciences, Palo Alto, CA). Excitation was 488 nm, and the emission filters used were 530 nm for Alexa 488 and 620 nm for PI. The
PI fluorescence signal (fluorescence pulse area versus pulse
width) was used to exclude doublets and aggregates from analysis. The
percentage of cells in the S, G0/G1, and
G2-M regions were determined with 20,000 cells.
Immunofluorescent Detection of Phosphorylated Histone
H3--
Cells were seeded at 4 × 105 cells/150-mm
dishes and incubated with or without 1 µM Cr(VI) for
24 h and then washed and maintained in complete medium. The cells
were harvested at various time points after treatment (1-7 days) and
fixed in 70% ethanol at Chromium Uptake--
Cells were seeded at 1 × 106 cells/60-mm dishes and incubated for 24 h prior to
sodium chromate exposure. One extra dish of each cell type was seeded
for determining the final cell number. Sodium chromate was prepared as
above and spiked with
Na251CrO4 (ICN, Irvine, CA).
Cells were treated with a final concentration of 3 µM
sodium chromate for 5 h at 37 °C. After sodium chromate treatment, cells were washed twice with PBS and lysed in 50 mM NaOH for 15 min. Cells were collected and combined with
5 ml of Ecolite scintillation mixture (ICN). Cell-associated
radioactivity was determined on a Beckman LS3801 scintillation counter.
Data were normalized to cell number.
Kinase Activity of ATM Was Enhanced after Cr(VI)
Treatment--
The lack of ATM protein in the ATM p53 Ser-15 Phosphorylation Was Delayed in
ATM Cr(VI)-induced Chk2 Thr-68 Phosphorylation Is
ATM-dependent--
Chk2 was found to phosphorylate Ser-20
on p53, and it is required for increased stability of p53 in response
to IR (34). Phosphorylation and activation of Chk2 have been shown to
be ATM-dependent in response to IR, whereas Chk2
phosphorylation is ATM-independent when cells are exposed to UV or
hydroxyurea (35). To investigate whether Cr(VI) exposure can induce
Chk2 phosphorylation and whether this phosphorylation is
ATM-dependent, we studied the Chk2 Thr-68 phosphorylation
in ATM+/+ (GM03440) and ATM ATM Contributes to Cr(VI)-induced Apoptosis--
ATM is
responsible for sensing and effecting certain DNA damage response
pathways that converge on p53 (33). Cr(VI)-induced apoptosis has been
shown to depend on activation of p53 (29). We used cells with
heterozygous or homozygous deletions in the ATM gene and studied their
sensitivity to Cr(VI)-induced apoptosis using the PS translocation
assay. As shown in Fig. 5A,
the basal apoptotic level in the untreated cells was around 2% in
ATM+/+ and ATM+/ ATM ATM Is Not Required for Cr(VI)-induced Growth Arrest, but It Is
Required for Cells to Recover from Cr(VI)-induced Growth
Arrest--
The single time point determinations of Cr(VI)-induced
apoptosis could not explain the markedly increased sensitivity of
ATM ATM-deficient Cells Fail to Exhibit Checkpoint Arrest in Early
Response to Cr(VI) Exposure, but Showed Latent Blockage at S and M
Phase Entry at Later Times--
To understand fully the nature of
Cr(VI)-induced growth arrest in ATM+/+ and ATM ATM kinase has been shown to be activated in response to DNA DSBs,
which can be induced by IR, radiomimetic agents, and during recombinational repair, but not to UVB/UVC or base-damaging agents (7),
indicating that ATM may act specifically in response to DSBs. However,
the existence of various abnormalities in AT patients other than
hypersensitivity to IR, such as premature aging, cancer predisposition,
and progressive cerebellar degeneration, has raised the possibility
that ATM may be involved in cellular processes in addition to the DSB
response (5). This has been supported by recent reports, which showed
the activation of ATM kinase by t-butylhydroperoxide
(15), CdCl2 (16), UVA (17), heat shock (18), insulin (19),
and MNNG (20). Furthermore, a recent study by Bakkenist et
al. (38) suggested that the activation of ATM might be the result
of DNA damage-induced changes in chromatin structure. In this study, we
showed enhanced ATM kinase activity in ATM+/+ cells after Cr(VI)
treatment, using p53 as a substrate (Fig. 2). Furthermore, the
respective phosphorylation of p53 Ser-15 and Chk2 Thr-68, which have
been shown to be physiological targets of ATM after DNA damage (6, 28),
was either delayed or abrogated in ATM-deficient cells after Cr(VI)
exposure (Figs. 3 and 4). Therefore, the results of the present study
suggest that Cr(VI) also activates ATM kinase despite no direct
evidence for Cr(VI) DSB induction.
Cr(VI) exposure results in a broad spectrum of genetic damage. One
lesion in particular, Cr-DNA interstrand cross-links (Cr-DDC) could
potentially contribute to the cytotoxicity of Cr(VI) (22) and may lead
to the activation of ATM. Our recent studies suggest that
recombinational repair is critical for the processing of potentially
lethal genetic lesions resulting from the intracellular reduction of
Cr(VI) (39). Indeed, studies by Morrison et al. (40) on
recombinational repair in AT cells confirmed the central role of ATM in
homologous recombinational repair of DNA damage. Misrepair of the
recombining DNA appears to be a feature of recombination in AT cells
(41). Therefore, the activation of ATM kinase by Cr(VI) may be the
result of (i) ATM directly sensing Cr-DDC or other lesions or (ii)
chromatin structure changes induced by Cr(VI) or (iii) ATM sensing DSBs
generated during recombinational repair of DNA-DNA cross-links. The
last hypothesis is supported by recent studies with MNNG, which
suggested that activation of ATM by the alkylating agent MNNG was
caused by the accumulation of strand breaks induced by the
activity of repair-associated endonucleases (20). Alternatively, ATM
has been reported to be a sensor of oxidative damage. Indeed,
deregulation of the oxidative stress response is part of the A-T
phenotype (16). Some studies have shown reactive oxygen species
generated during Cr(VI) reduction; however, the experiments were
conducted using extraordinarily high Cr(VI) doses (100 µM-2 mM) and already malignant cells
(42-46). The doses employed in this study (0.1-6 µM)
have not been shown to produce reactive oxygen species and are more
relevant to human exposure.
ATM has been implicated in regulating phosphorylation and
induction of p53 in cells exposed to IR through several
mechanisms (47). ATM induces p53 by directly phosphorylating Ser-395 of murine double minute clone 2 protein (MDM2) (48), which decreases the
ability of MDM2 to shuttle p53 from the nucleus to the
cytoplasm, and by indirectly phosphorylating Ser-20 of p53 through the
activation of Chk2 (35) (34). ATM is also implicated in the direct
phosphorylation of Ser-15 of p53 (33) (7). Although the latter
phosphorylation has little effect on the half-life of p53 or on its
ability to cause cell cycle arrest or apoptosis, it has been suggested
that phosphorylation of p53 Ser-15 might facilitate other
post-translational modifications of p53, such as acetylation of
Lys-382, which in turn facilitates the sequence-specific DNA
binding (47). Indeed, poor p53 induction has been found in
ATM-deficient cells after IR exposure (32). We have shown previously
that p53 is activated in normal human fibroblasts in response to Cr(VI)
exposure (28). After Cr(VI) treatment of human lung epithelial A549
cells, p53 protein became phosphorylated at Ser-15 and acetylated at
Lys-382, whereas MDM2 protein was dissociated (49). Our
additional studies showed that Cr(VI) exposure was accompanied by
induction of both p53-transactivated and p53-independent proapoptotic
genes in human lung fibroblasts (50). Here, we show that p53 Ser-15 and
Chk2 Thr-68 phosphorylations were either delayed or abrogated in
ATM-deficient cells (Figs. 3 and 4). The phosphatidylinositol
3-kinase-related kinases other than ATM, i.e. DNA-PK and
ATR, are implicated in DNA repair and signal transduction
pathways induced by DSBs. DNA-PK is implicated in nonhomologous
end-joining repair (6), whereas ATR is involved in the later
stages of the cellular response to DSBs (51). These other
phosphatidylinositol 3-kinase-related kinases share many targets that
are phosphorylated by ATM following DSBs (52). This functional
redundancy may explain the delayed phosphorylation of p53 Ser-15 in
ATM-deficient cells in response to Cr(VI).
Studies by us and others have shown that Cr(VI)-induced apoptosis is
p53-dependent (29, 53). Indeed, Cr(VI)-induced apoptosis in
human lung fibroblast cells is dependent on the presence of p53, which
is activated in response to Cr(VI) exposure (28, 29). Cr(VI) was shown
to increase p53 protein stability in human lung fibroblast cells, by a
4-6-fold increase in p53 protein levels, and was accompanied by p53
translocation to the nucleus (29). The p53 requirement for
Cr(VI)-induced apoptosis was confirmed in two models: (i) mouse
dermal fibroblasts heterozygous and homozygous for p53 deletion, and
(ii) human lung fibroblast cells transiently transfected with the human
papilloma virus E6 gene, which targets p53 for degradation, thus
creating a functional p53 deletion (28). Previous studies have shown
that IR-induced apoptosis was reduced in AT cells compared with normal
cells (54), and the failure to undergo apoptosis has been associated
with an altered pattern of p53 induction after IR exposure (55). In the
present study, ATM-deficient cell lines were significantly resistant to
Cr(VI)-induced apoptosis (Fig. 5), which might be because of the
attenuated p53 induction after Cr(VI) exposure. These results indicate
that ATM is a major upstream activator of p53-dependent
Cr(VI)-induced apoptosis.
The altered induction of p53 does not explain AT radiosensitivity. It
has been shown by numerous investigators that ATM-deficient cells are
hypersensitive to IR-induced clonogenic lethality. Shackelford et
al. (15) showed that GM03395 ATM The population-wide cell cycle arrest in response to DNA damage
provides the opportunity for a cell to regulate its own proliferation, thereby avoiding the propagation of damaged DNA. Depending on the
extent of the genotoxic insult, an arrested cell could either regain
its replicative potential by repairing the damaged DNA or be removed
from the dividing population by undergoing either terminal growth
arrest or apoptosis (56, 57). Studies by us and numerous investigators
have shown that ATM-deficient cell lines are impaired in their
activation of cell cycle checkpoints (Fig. 8) (5), which suggests that
ATM-deficient cells may lose the opportunity to regulate proliferation,
thereby causing the propagation of damaged DNA. Furthermore, the ATM
gene is involved in the DNA damage repair pathway by phosphorylating
Brca1 (8), c-Abl (10), NBS1 (8), Gadd45 (55), etc. Collectively, the deficiency in cell cycle checkpoint arrest and DNA damage repair may
contribute to the genetic instability of ATM-deficient cells. Indeed,
ATM-deficient cells exhibit higher numbers of chromosomal gaps and
breaks that persist for longer periods of time after IR (2) (58).
Therefore, the accumulation of unrepaired damaged DNA may contribute to
the hypersensitivity to Cr(VI)-induced clonogenic lethality observed in
ATM-deficient cell lines. In the present study we showed that
Cr(VI)-exposed normal human fibroblasts undergo an extended, but not
terminal, form of growth arrest whereas they presumably slowly repaired
the damaged DNA and recovered from this growth arrest after a
considerable period. This is supported by our previous studies (30),
which also found a similar transient cell growth arrest followed by
replicative growth recovery in telomerase-immortalized normal human
fibroblasts after a 24-h exposure to 1 µM Cr(VI), a
concentration at which no apoptosis was detected. In contrast,
ATM-deficient cells were unable to recover from this growth arrest.
Cell cycle analysis of ATM+/+ and ATM/
cells
were markedly resistant to Cr(VI)-induced apoptosis but considerably
more sensitive to Cr(VI)-induced clonogenic lethality than wild type
cells, indicating that resistance to Cr(VI)-induced apoptosis did not
confer a selective survival advantage. However, analysis of long term
growth arrest revealed a striking difference: ATM
/
cells were
markedly less able to recover from Cr(VI)-induced growth arrest. This
indicates that terminal growth arrest is the fate of these
apoptosis-resistant cells. In summary, ATM is involved in cellular
response to a complex genotoxin that may not directly induce DSBs. Our
data suggest that ATM is a major signal initiator for genotoxin-induced
apoptosis but, paradoxically, also contributes to maintenance of cell
survival by facilitating recovery/escape from terminal growth arrest.
The results also strongly suggest that terminal growth arrest is not merely an extended or even irreversible form of checkpoint arrest, but
instead an independent and unique cell fate pathway.
INTRODUCTION
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
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DISCUSSION
REFERENCES
/
cells were
resistant to Cr(VI)-induced apoptosis compared with wild type
cells. However, ATM
/
cells were more sensitive to Cr(VI)-induced
clonogenic lethality than wild type cells, suggesting that the
resistance to Cr(VI)-induced apoptosis in ATM
/
cells did not confer
a selective survival advantage. Although ATM
/
cells exhibited a
similar sensitivity to Cr(VI)-induced growth arrest, they were markedly
less able to recover from this growth arrest than ATM+/+ cells. These
results indicate that the ATM gene is required for genotoxin-induced
apoptosis and also contributes to maintenance of cell survival by
facilitating recovery and/or escape from terminal growth arrest.
EXPERIMENTAL PROCEDURES
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-32P]ATP (ICN, Irvine, CA) and
1 µg of GST- p531-101 (generous gift of Dr.
Michael Kastan, St. Jude Children's Research Hospital, Memphis, TN)
and incubated for 30 min at 30 °C. The reaction was stopped by the
addition of SDS-PAGE loading buffer. The proteins were
electrophoretically separated by SDS-PAGE, transferred to polyvinylidene difluoride membranes, and exposed to x-ray film. p53
Western blots (Ab-6 from Oncogene Research Products, Boston, MA) were
conducted on the same membranes after decay of 2 half-lives to confirm
the equal amount of GST-p531-101 substrate used in the
reaction. The relative quantity of immunoreactive and
32P-labeled proteins was determined with a Personal
Densitometer SI (PDSI) and ImageQuant software (Molecular Dynamics,
Sunnyvale, CA).
20 °C. The cells were resuspended in
0.25% Triton X-100 in PBS and incubated on ice for 15 min. The cells
were pelleted by centrifugation and resuspended in PBS containing 1%
bovine serum albumin with 0.25 µg/ml of a polyclonal antibody that
specific recognizes the phosphorylated form of histone H3 (Upstate
Biotechnology, Lake Placid, NY) and incubated for 3 h at room
temperature. The cells were then rinsed with PBS containing 1% bovine
serum albumin and incubated with Alexa 488-conjugated goat anti-rabbit
secondary antibody (Molecular Probes) diluted at a ratio of 1:100 in
PBS containing 1% bovine serum albumin. The cells were then stained
with PI and analyzed using a FACSort as described above for BrdUrd and
PI. The percentage of cells containing phosphorylated histone H3,
undergoing mitosis, was determined with 20,000 cells.
RESULTS
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
/
and mutant cell
lines was confirmed by immunoblotting analysis. As expected, the ATM+/+ and ATM+/
cell lines contained similar levels of ATM protein (Fig.
1). It has been shown that ATM isolated
from cells exposed to IR exhibits an increased ability to phosphorylate
p53 in vitro on a single residue, Ser-15, in response to IR
(7). To investigate whether the kinase activity of ATM was
enhanced after exposure of cells to a complex genotoxin, we measured
ATM kinase activity in ATM+/+ and ATM
/
cell lines after a 3-h
treatment with 0 or 6 µM Cr(VI). ATM immunoprecipitates
were used to phosphorylate GST-p531-101 in
vitro. ATM protein kinase activity toward GST-p531-101, expressed as the amount of
GST-p531-101 phosphorylated by ATM normalized to the total
GST-p531-101, was increased from 0.36 to 0.69 (Fig.
2B) (p < 0.05). This increased kinase activity is attributed specifically to the
activation of ATM protein. No ATM-immunoreactive protein was
present when preimmune serum was used for immunoprecipitation.
Furthermore, no protein kinase activity was found in immunoprecipitates
from ATM-deficient cells or in Cr(VI)-treated ATM+/+ cells
immunoprecipitated with preimmune serum (data not shown). Therefore,
the ATM kinase appears to be activated in response to Cr(VI) treatment,
and it phosphorylates p53 in vitro.
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Fig. 1.
ATM status. Total cellular protein was
extracted from normal fibroblasts (GM03440) and fibroblasts with either
heterozygous (GM03397) or homozygous ATM deletions (GM03395 and
GM02052). The protein was separated by SDS-PAGE, and ATM protein was
detected by immunoblotting.
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Fig. 2.
Phosphorylation of GST-p531-101
by ATM in vitro. A, normal fibroblasts
(GM03440) and fibroblasts with a homozygous ATM deletion (GM03395) were
treated with 0 or 6 µM Cr(VI) for 3 h. ATM was then
immunoprecipitated (A, bottom panel) and used in
an in vitro kinase assay with [ -32P]ATP and
GST-p531-101 as substrates. Proteins from each reaction
were separated by SDS-PAGE, transferred to polyvinylidene difluoride
membranes, and exposed to x-ray film (A, top
panel). Equal amounts of GST-p531-101 used in each
reaction were confirmed by immunoblotting for p53 (A,
middle panel). B, phosphorylation of
GST-p531-101 by ATM in vitro. Data were
expressed as a ratio of GST-p531-101 phosphorylated by ATM
to total GST-p531-101. Results are the means of three
independent experiments. Bars, S.E. * indicates a
statistically significant increase from the control (p < 0.05).
/
Cells--
A delay in phosphorylation of p53
Ser-15 has been shown in ATM-deficient cells in response to IR,
indicating that Ser-15 is a physiological target of ATM after DNA
damage (32, 33). To examine this effect in response to a complex
genotoxin, we studied the expression of p53 Ser-15 phosphorylation in
ATM+/+ and ATM
/
cells after 0, 1, 3, 6, 16, and 24 h of
continuous exposure to 6 µM Cr(VI). As shown in Fig. 3,
A and C, a 3.5-fold increase in phosphorylation of p53 Ser-15 was observed within 1 h
after 6 µM Cr(VI) treatment in ATM+/+ cells. In contrast, no phosphorylation of p53 Ser-15 was detected within 1 h in
ATM
/
cells. After 3, 6, 16, and 24 h of treatment,
respectively, the phosphorylation of p53 Ser-15 increased 10-, 6-, 12-, and 37-fold in ATM+/+ cells, but only 2.3-, 2.8-, 7.2-, and 13-fold in
ATM
/
cells. Furthermore, we studied the induction of p53 Ser-15
phosphorylation in ATM+/
and ATM mutant cells after 0, 1, 3, and
24 h of continuous exposure to 6 µM Cr(VI). Similar
to ATM+/+ cells, the phosphorylation of p53 Ser-15 increased 3-, 10-, and 27-fold in ATM+/
cells after 1, 3, and 24 h of Cr(VI)
treatment, but only 1.4-, 4-, and 24-fold in ATM mutant cells (Fig.
3B). Furthermore, to test whether ATM activation can be
observed with Cr(VI) concentrations as low as 1 µM, we
studied the expression of phospho-p53 Ser-15 in ATM+/+ and ATM
/
cells after 1 µM Cr(VI) treatment for 3 h. A
2.3-fold increase in phosphorylation of p53 Ser-15 was observed in
ATM+/+ cells, but only 1.1-fold in ATM
/
cells (data not shown).
Taken together, these results show that p53 phosphorylation is delayed significantly in ATM
/
cells.
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Fig. 3.
p53 Ser-15 phosphorylation in ATM+/+ and
ATM /
cells after Cr(VI)
exposure. A, normal fibroblasts (GM03440) and
fibroblasts with homozygous ATM deletions (GM03395) were incubated with
6 µM Na2CrO4 for 1, 3, 6, 16, and 24 h, and total cellular protein was extracted after the
treatment. Proteins were separated by SDS-PAGE, and p53 Ser-15
phosphorylation was detected by immunoblotting (A,
upper panel). The same amount of protein loading was
confirmed by immunoblotting for G3PDH (A, lower
panel). B, fibroblasts with heterozygous ATM deletions
(GM03397) and ATM mutant fibroblasts (GM02052) were treated with 6 µM Na2CrO4 for 1, 3, and
24 h, and total cellular protein was extracted after the
treatment. Proteins were separated by SDS-PAGE, and p53 Ser-15
phosphorylation was detected by immunoblotting (B,
upper panel). The same amount of protein loading was
confirmed by immunoblotting for G3PDH (B, lower
panel). C, p53 Ser-15 phosphorylation in ATM+/+ and
ATM
/
cells after Cr(VI) exposure. Data are the means ± S.E.
of three independent experiments and are expressed as -fold of the
respective control, normalized to G3PDH, in the absence of Cr(VI). *
indicates a statistically significant difference between ATM+/+ cells
and ATM
/
cells at p < 0.05.
/
(GM03395) cells after 0, 30, and 60 min of continuous exposure to 6 µM Cr(VI). As shown in
Fig. 4, A and B, a
2-fold increase of phosphorylation of Chk2 Thr-68 was observed within
30 min after a 6 µM Cr(VI) treatment in ATM+/+ cells,
whereas no increased phosphorylation of Chk2 Thr-68 was detected within
30 min of Cr(VI) exposure in ATM
/
cells. After a 60-min treatment,
the phosphorylation of Chk2 Thr-68 increased 2.4-fold in ATM+/+ cells,
but again, no phosphorylation was detected in ATM
/
cells.
Furthermore, total Chk2 expression, as determined by Western blotting,
was not changed after Cr(VI) exposure because equal levels of Chk2 were
found before and after Cr(VI) treatment in both ATM+/+ and ATM
/
cells (Fig. 4A). Therefore, the results show that Cr(VI)
exposure induces phosphorylation of Chk2 Thr-68, and this
phosphorylation is ATM-dependent.
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Fig. 4.
Chk2 Thr-68 phosphorylation in ATM+/+ and
ATM /
cells after Cr(VI)
exposure. A, normal fibroblasts (GM03440) and
fibroblasts with homozygous ATM deletions (GM03395) were incubated with
6 µM Na2CrO4 for 0, 30, and 60 min. Total cellular protein was extracted after the treatment.
Proteins were separated by SDS-PAGE, and Chk2 Thr-68 phosphorylation
and total Chk2 were detected by immunoblotting (A,
top and middle panels). The same amount of
protein loading was confirmed by immunoblotting for G3PDH
(A, bottom panel). B, Chk2 Thr-68
phosphorylation in ATM+/+ and ATM
/
cells after Cr(VI) exposure.
Data are the means ± S.E. of three independent experiments and
are expressed as -fold of the respective control, normalized to G3PDH,
in the absence of Cr(VI). * indicates a statistically significant
difference between ATM+/+ cells and ATM
/
cells at p < 0.05.
cells but was 2-3-fold higher in ATM
/
cells.
Treatment with 3 µM Cr(VI) caused a modest increase in
apoptosis in both ATM+/+ and ATM+/
, but not ATM
/
cells. Treatment
with 6 µM Cr(VI) induced apoptosis in 40% of both ATM+/+
and ATM+/
cells, but the percentage of apoptosis (17-23%) was
significantly lower in ATM
/
cells. When expressed as a -fold of
untreated control, treatment with 3 µM Cr(VI) induced a
2.6-4.3-fold increase in apoptosis in both ATM+/+ and ATM+/
cells
but only a 1.3-fold increase in ATM
/
cells. ATM+/+ and ATM+/
cells exhibited a 20-fold increase in apoptosis after 6 µM Cr(VI) exposure, whereas ATM
/
cells showed marked
resistance to Cr-induced apoptosis (p < 0.05).
Furthermore, we confirmed the results of PS translocation assay by
studying the cellular level of cleaved caspase-3. Caspase-3 is one of
the key executioners of apoptosis. Activation of caspase-3 requires proteolytic processing of its inactive zymogen into activated P12 and P17 subunits (36). Western blotting with an antibody to cleaved
caspase-3 showed that treatment with 6 µM Cr(VI) produced a significant 8-fold induction of cleaved caspase-3 in ATM+/+ cells
(p < 0.05) but no change in ATM
/
cells (Fig. 5,
B and C). The results of these studies suggest
that ATM is required for the majority of Cr(VI)-induced apoptosis. We
also explored the possibility that the decreased apoptotic response of
the ATM
/
cell lines to Cr(VI) exposure may be caused by an impaired
Cr(VI) uptake mechanism. Therefore, we studied the uptake of
Na251CrO4 at a concentration
of 3 µM for 5 h in all cell lines studied. The
average uptake was 2.5 nmol/106 cells, with no difference
among the different cell lines (data not shown).
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Fig. 5.
Resistance of
ATM /
cells to
Cr(VI)-induced apoptosis. A, normal fibroblasts
(GM03440) and fibroblasts with either heterozygous (GM03397) or
homozygous ATM deletions (GM03395 and GM02052) were incubated with 3 or
6 µM Na2CrO4 for 24 h
followed by culture in complete medium without
Na2CrO4 for an additional 24 h.
Apoptosis was assessed as the proportion of cells with translocated
phosphatidylserine, as determined by binding of fluorescent annexin V. Data are expressed as a percent of apoptotic cells and are the
means ± S.E. of three independent experiments. * indicates a
statistically significant difference between ATM+/+ cells and ATM
/
cells at p < 0.05. B, normal fibroblasts
(GM03440) and fibroblasts with homozygous ATM deletions (GM03395) were
incubated with 3 or 6 µM
Na2CrO4 for 24 h followed by
culture in complete medium without
Na2CrO4 for an additional 24 h.
Total cellular protein was extracted after the treatment. Proteins were
separated by SDS-PAGE, and cleaved caspase-3 was detected by
immunoblotting (B, upper panel). The same amount
of protein loading was confirmed by immunoblotting for G3PDH
(B, lower panel). C, Cr(VI)-induced
cleaved-caspase 3 expression. Data are the means ± S.E. of three
independent experiments and are expressed as -fold of the respective
control, normalized to G3PDH, in the absence of Cr(VI). * indicates a
statistically significant difference from the control cells at
p < 0.05.
/
Cells Are Hypersensitive to Cr(VI)-induced
Clonogenic Lethality--
Because the lack of ATM conferred resistance
to Cr(VI)-induced apoptosis, we determined clonogenic survival after
Cr(VI) exposure in ATM+/+, ATM+/
, and ATM
/
cells. Clonogenicity
is an indicator of long term cell survival and replicative potential
after exposure to a genotoxic agent. By determining the clonogenicity
of Cr(VI)-exposed cells we were able to examine the cumulative effect
of Cr(VI)-exposure on a cell population. The results are shown in Fig.
6 and are expressed as the percentage of
the respective control, i.e. in the absence of Cr(VI). The
ATM
/
cells showed significantly less clonogenic survival after 0.1, 0.5, or 1 µM Cr(VI) treatment compared with ATM+/+ and
ATM+/
cells (p < 0.05). The percent clonogenic survival for 0.1, 0.5, and 1 µM Cr(VI) was 98, 78, and
51-59% for ATM+/+ and ATM+/
cells compared with 60-84, 28-51,
7-14% for the two ATM-deficient cell lines, respectively. Therefore, despite the resistance to Cr(VI)-induced apoptosis, these results indicate that lack of the ATM gene results in increased clonogenic lethality.
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Fig. 6.
Effect of the ATM gene on clonogenic survival
after exposure to Cr(VI). Normal fibroblasts (GM03440) and
fibroblasts with either heterozygous (GM03397) or homozygous ATM
deletions (GM03395 and GM02052) were incubated with 0.1-1
µM Na2CrO4 for 24 h
and were washed and reseeded to allow for colony formation. Colonies
were stained with crystal violet and counted 12 days later.
Inset, clonogenicity data plotted on a log scale. The data
are expressed as percentage of respective control, in the absence of
Cr(VI), and are the means ± S.E. of three independent
experiments. * indicates a statistically significant difference from
the control cells at p < 0.05.
/
cells to Cr(VI)-induced clonogenic lethality. Therefore, we
examined both ATM+/+ (GM03440) and ATM
/
cell (GM03395) growth by
counting the cell number over a 16-day period in populations of cells
exposed to 0-1 µM Cr(VI) for 24 h. Data were
expressed as -fold change compared with untreated controls (Fig.
7). In both cell lines, Cr(VI) induced a
similar dose-dependent decrease in cell growth, which
lasted up to 8 days after treatment. However, after 8 days a striking
difference emerged. The Cr(VI)-treated ATM+/+ cells recovered from the
decreased cell growth and caught up with the nontreated cells at day
12, whereas the number of 0.5 µM and 1 µM
Cr(VI) treated-ATM
/
cells remained at 70, and 41% of the nontreated control even 16 days after exposure (Fig. 7). We further explored the possibility that the persistent decreased number of
ATM
/
cells after Cr(VI)-exposure may be due, in part, to a low
level of cell death induced by Cr(VI). Therefore, we studied apoptotic
cell death by PS translocation assay at days 1, 8, 12, and 16 after
24 h 1 µM Cr(VI) exposure in ATM+/+ and ATM
/
cells. Our results showed that no apoptotic cell death was
induced by 1 µM Cr(VI) in either ATM+/+ or ATM
/
cells
at any time point. The respective proportion of apoptotic cells after 1 µM Cr(VI) exposure ranged from 0.9- to 1.1-fold of the
nontreated control in ATM+/+ cells and from 0.8- to 1.2-fold in
ATM
/
cells (data not shown). The results indicate that the
decreased cell number observed in ATM
/
cells was caused by growth
arrest and not apoptotic cell death induced by Cr(VI) treatment. These
data strongly indicate that the initial growth arrest after Cr(VI)
treatment is not dependent on ATM; however, ATM is either required for
cells to recover from Cr(VI)-induced growth arrest or for preventing
the growth arrest from becoming terminal.
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Fig. 7.
Response of ATM+/+ and
ATM /
cells to
Cr(VI)-induced growth arrest. Normal fibroblasts (GM03440) and
fibroblasts deficient in the ATM gene (GM03395) were incubated with 0, 0.1 (A), 0.5 (B), or 1 µM
(C) Na2CrO4 for 24 h,
and cell numbers were counted at 1-6, 8, 12, or 16 days after the
Na2CrO4 removal. Data are the means ± S.E. of three independent experiments and are expressed as a percent
of the respective control in the absence of Cr(VI). *
indicates a statistically significant difference between ATM+/+ cells
and ATM
/
cells at p < 0.05.
/
cells, we evaluated
the G1/S and G2/M checkpoint transitions in
ATM+/+ and ATM
/
cells at days 1, 2, 4, and 7 after 24-h 1 µM Cr(VI) exposure. G1 checkpoint function, as reflected by delay or suppression of entry into S phase, was analyzed by BrdUrd and PI double staining (Fig.
8). Exposure of ATM+/+ cells to 1 µM Cr(VI) resulted in a suppression of S phase entry, as
measured by flow cytometry. The percentage of S phase cells decreased
to 53% of that of the nontreated control at day 1. By day 7, ATM+/+
cells were released from the G1 checkpoint and reentered S
phase, as the S phase population increased to 73% of nontreated
control. When ATM
/
cells were subjected to the same treatment,
comparatively little inhibition of S phase entry was observed at day 1, as S phase population remained at 88% of the untreated control.
However, at day 7, ATM
/
cells were markedly less able to enter S
phase, as the S phase population decreased to 42% of that in the
untreated control. G2/M checkpoint function, as reflected
by suppression of entry into mitosis, was measured by flow cytometric
assessment of histone H3 phosphorylation. Histone H3 is cyclically
phosphorylated during mitosis (37). After Cr(VI) exposure, ATM+/+ cells
were blocked from entering mitosis at day 1 (data not shown); however,
these cells were released from the G2 checkpoint by day 7, as the mitotic population increased from 1.6% of the total
G2/M population at day 4 to 4.1% at day 7. In contrast,
ATM
/
cells were unable to enter mitosis at day 7, as the mitotic
cells remained at 0.6% of G2/M population. Therefore, despite the deficiency of checkpoint arrest in the early response to
Cr(VI), these results suggest that ATM
/
cells were arrested and
impaired in the ability to enter both S and M phase at later times
after Cr(VI) exposure.
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Fig. 8.
Flow cytometric analysis of G1
checkpoint arrest in response to Cr(VI) in ATM+/+ and
ATM /
cells. Normal
fibroblasts (GM03440) and fibroblasts deficient in the ATM gene
(GM03395) were incubated with 0 or 1 µM
Na2CrO4 for 24 h, and cells were
incubated with BrdUrd for 1 h before collection at various time
points. Cells were fixed and incubated with mouse anti-BrdUrd antibody
and Alexa 488-conjugated goat anti mouse secondary antibody. Cells were
then stained with PI. Dot plots show incorporation of BrdUrd
into DNA (y axis) as an indication of DNA synthesis and PI
fluorescence (x axis) as an indication of DNA content.
Numbers indicate the percentage of S phase population after
Cr(VI) exposure and are expressed as a percent of the respective
control in the absence of Cr(VI).
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
/
and GM02052 ATM mutant cells
exhibit increased sensitivity to IR-induced clonogenic lethality compared with normal human fibroblasts. Furthermore, using
neocarzinostatin (generous gift of Dr. Irving Goldberg, Harvard Medical
School), a radiomimetic agent, we found that 10 and 25 ng/ml
neocarzinostatin caused 82 and 35% clonogenic survival in GM03440
ATM+/+ cells, but only 40 and 7% in GM03395 ATM
/
cells (data not
shown). Indeed, in primary fibroblasts, which exhibit an attenuated
degree of apoptosis after a genotoxic treatment, the difference in
radiosensitivity between ATM-deficient cells and normal cells is still
observed (12). Here, we showed that ATM-deficient cells were
hypersensitive to Cr(VI)-induced clonogenic lethality (Fig. 6), which
indicates that the resistance to Cr(VI)-induced apoptosis in
ATM-deficient cells did not confer a selective survival advantage.
/
cells showed that ATM+/+
cells undergo Cr(VI)-induced G1/S and G2/M
checkpoint arrest (Figs. 8 and 9), which
presumably allows an opportunity for DNA repair before replication. In
contrast, ATM
/
cells failed to activate checkpoints in early
response to Cr(VI) (Fig. 8). Therefore, the combination of the failure to activate checkpoints and the deficiency in DNA damage repair jointly
contribute to the accumulation of unrepaired DNA damage in
ATM-deficient cells. This, in turn, causes ATM
/
cells with accumulated unrepaired DNA damage to be removed from the proliferative population by undergoing terminal growth arrest. This is supported by
our data, which showed that ATM+/+ cells resumed proliferation at later
time points; however, ATM
/
cells remained in an arrested state in
which few ATM
/
cells were able to enter S or M phase. Therefore,
these results indicate that terminal growth arrest is the fate of these
apoptosis-resistant cells, and ATM is required for recovery from
genotoxin-induced cell growth arrest.
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Fig. 9.
Flow cytometric analysis of G2/M
checkpoint arrest in response to Cr(VI) in ATM+/+ and
ATM /
cells.
A, normal fibroblasts (GM03440) and fibroblasts deficient in
the ATM gene (GM03395) were incubated with 0 or 1 µM
Na2CrO4 for 24 h. Cells were then
collected at various time points and fixed. Cells in mitosis were
determined by staining with an antibody to phosphohistone H3, followed
by Alexa 488-conjugated secondary antibody. Contour plots show staining
of phosphohistone H3 as an indication of mitotic cell population
(y axis) and PI fluorescence (x axis) as an
indication of DNA content. B, G2/M checkpoint
transition in response to Cr(VI) in ATM+/+ and ATM
/
cells. Data are
the means ± S.D. and are the mitotic cells expressed as a percent
of total G2/M phase population.
In summary, our data suggest that ATM is activated after Cr(VI)
exposure, and ATM is a major signal initiator for Cr(VI)-induced apoptosis. Paradoxically, ATM is also necessary for clonogenic survival, specifically for the cells to recover from the protracted growth arrest or escape from the terminal growth arrest induced by
Cr(VI). We propose a model for the role of the ATM gene in the cellular
response to a complex genotoxin (Fig.
10). Exposure of ATM+/+ cells to a
complex genotoxin, such as Cr(VI), will cause the activation of the ATM
protein (Fig. 10A), which causes the majority of the cells
to undergo a transient (albeit protracted) cell cycle checkpoint arrest
by activating downstream proteins such as p53 and ChK2, presumably to
allow an opportunity for DNA repair before replication. Cells in which
DNA repair is complete will regain replicative potential, whereas cells
in which DNA repair is incomplete will be eliminated from the cell
cycle by apoptosis or possible terminal cell growth arrest. In
contrast, because of the lack of the ATM gene, ATM-deficient cells fail to activate cell cycle checkpoint arrest after complex genotoxin insult
(Fig. 10B). The majority of the cells undergo terminal
growth arrest. A stochastic fraction of cells may survive with
unrepaired DNA damage and exhibit a predisposition to genomic
instability and neoplasia. Taken together, this paradigm also strongly
suggests that terminal growth arrest is not merely an extended or
irreversible form of checkpoint arrest, but instead an independent and
unique cell fate pathway.
|
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ACKNOWLEDGEMENTS |
---|
We thank Dr. Michael Kastan for the GST p531-101 fragment used in this study and Dr. Irving Goldberg for the neocarzinostatin. We also thank Dr. Stephanie Constant for help in cell cycle data analysis, Drs. Daryl Pritchard and Travis O'Brien for helpful discussions, and Fatema Rafiqi and Carla Zingariello for technical assistance.
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FOOTNOTES |
---|
* This work was supported by National Institutes of Health Grants ES05304 and ES09961 (to S. R. P.).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.
This work was conducted in partial fulfillment of the requirements
for a doctorate of philosophy in molecular and cellular oncology,
Columbian Graduate School of Arts and Sciences, The George Washington University.
§ To whom correspondence should be addressed: Dept. of Pharmacology, The George Washington University Medical Center, 2300 I St. N.W., Washington, D. C. 20037. Tel.: 202-994-3286; Fax: 202-994-2870; E-mail: phmsrp@gwumc.edu.
Published, JBC Papers in Press, March 10, 2003, DOI 10.1074/jbc.M210560200
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ABBREVIATIONS |
---|
The abbreviations used are: AT, ataxia telangiectasia; ATM, ataxia telangiectasia mutated; BrdUrd, bromodeoxyuridine; CHAPS, 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonic acid; DSB(s), double strand break(s); G3PDH, glyceraldehyde-3-phosphate dehydrogenase; GST, glutathione S-transferase; IR, ionizing radiation; MNNG, N-methyl-N'-nitro-N-nitrosoguanidine; PBS, phosphate-buffered saline; PI, propidium iodide; PS, phosphatidylserine.
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