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INTRODUCTION |
Genistein is a naturally occurring isoflavenoid that is abundant
in soy beans and has been reported to have anti-carcinogenic effects
(1-3). Soy-based diets have been reported to reduce the risk of a
variety of cancers including, breast, prostate, and colon cancers
(4-6). Moreover, genistein has been reported to be responsible for low
rates of breast cancer in Asian women (7). However, the mechanism of
action of genistein on cells remains unclear. Genistein acts as a broad
specificity tyrosine kinase inhibitor (8) but also inhibits
topoisomerase II and generates DNA damage by stabilizing the covalent
topoisomerase II-DNA cleavage complex (9-11). Genistein has been
reported to induce DNA laddering and apoptosis in breast cancer cell
lines (10-14), suggesting that in some cells it initiates a DNA damage
response that leads to apoptosis. Other topoisomerase II poisons, such
as etoposide, induce apoptosis in a variety of cell lines (15-18).
Treatment of HL60 cells with etoposide induces caspase-mediated
cleavage of ATM (ataxia-telangiectasia
mutated protein; Ref. 19), and the catalytic subunit of the
DNA-dependent protein kinase (DNA-PKcs) (16-18,
20).1
DNA-PKcs and ATM are both members of a family of phosphatidylinositol
3-kinase-like proteins (reviewed in Refs. 21 and 22). ATM is mutated in
the human autosomal disorder, ataxia-telangiectasia (A-T), which is
characterized by ataxia, abnormal vasodilation, radiosensitivity,
predisposition to cancer, and immune defects (reviewed in Ref. 23). ATM
encodes a polypeptide of ~360 kDa that, like DNA-PKcs, contains a
C-terminal phosphatidyl inositol 3-kinase-like catalytic domain
(24-26). Another characteristic of A-T cells is failure to arrest at
G2/M following exposure to ionizing radiation (27, 28).
Indeed, ATM is required for activation of p53 and Chk2, resulting in
cell cycle arrest at G1/S and G2/M respectively
(reviewed in Ref. 29). Cells that lack ATM show delayed up-regulation
and phosphorylation of p53 (reviewed in Ref. 30) and defective
phosphorylation of Chk2 in response to ionizing radiation (31, 32).
In vitro, ATM is a manganese-dependent serine
threonine protein kinase that phosphorylates serine 15 of p53 (33-35)
and N-terminal sites in Chk2 (35). ATM is therefore a key player in
signaling DNA damage to cell cycle checkpoints.
Because etoposide and genistein have both been reported to induce
apoptosis by initiating topoisomerase II-induced DNA damage, we were
interested to determine whether they activated an
ATM-dependent pathway. Indeed, genistein has been shown to
induce up-regulation of p53 protein levels in ATM-positive cells (36).
Here we show that treatment of ATM-positive cells with genistein
activates the DNA-binding function of p53, induces phosphorylation of
p53 at serine 15, and induces phosphorylation of Chk2 at threonine 68. Genistein did not induce activation of p53 or phosphorylation of Chk2
in two ATM-deficient human cell lines, suggesting that genistein
activates a pathway that requires ATM or is mediated by ATM. Moreover,
we show that the topoisomerase inhibitor, etoposide, but not the
tyrosine kinase inhibitor herbimycin A, also induces phosphorylation of
p53 at serine 15 and phosphorylation of Chk2, supporting the idea that
genistein induces DNA damage that is recognized by an
ATM-dependent pathway. In contrast, etoposide induced
phosphorylation of both p53 and Chk2 in ATM-deficient cells as well as
in normal cells, suggesting that etoposide activates protein kinases
other than ATM in vivo.
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MATERIALS AND METHODS |
Reagents--
Genistein was obtained from Calbiochem. Etoposide
was purchased from Sigma. Herbimycin was purchased from Life
Technologies, Inc.
Cells--
ATM-positive cells (BT and C3ABR) and ATM-deficient
cells (L3 and AT1ABR) (37) were grown at 37 °C, 5% CO2
in RPMI containing 10% fetal calf serum (Hyclone). Genistein,
etoposide, and herbimycin A were dissolved in Me2SO and
stored as stock solutions at
20 °C. Where indicated genistein,
etoposide, or herbimycin A was added directly to the cell medium in
which the cells were suspended. Control cells were treated with an
equivalent volume of Me2SO. Where indicated, cells were
irradiated, in the presence of serum and medium, with 10 Gy ionizing
radiation using a Gammacell 1000 Cesium 137 source (MDS Nordion), at a
dose rate of 250 cGy/min. After irradiation or addition of
genistein, etoposide, or herbimycin A, cells were returned to a
humidified incubator at 37 °C under 5% CO2.
Protein Extracts--
Crude cytoplasmic (S10) and nuclear (P10)
extracts were made as described previously (38). Briefly, cells were
harvested and washed in phosphate buffered saline, followed by
hypotonic buffer (low salt buffer; Ref. 39). Cell pellets were
lysed by a single freeze thaw and centrifuged at 10,000 × g for 10 min to produce the S10 supernatant. The resulting
pellet was extracted with low salt buffer containing 0.5 M
NaCl, 10 mM MgCl2, 1 mM dithiothreitol, and the supernatant was recovered after centrifugation at 10,000 × g for 10 min to produce the P10. Protease
inhibitors (aprotinin, leupeptin, and phenylmethysulfonyl fluoride,
5 µg/ml, 5 µg/ml, and 0.2 mM, respectively) and
phosphatase inhibitors (sodium vanadate,
-glycerol phosphate and
sodium fluoride at 0.04 mM, 2 mM, and 10 mM, respectively) were added to cell extracts immediately
after freeze-thaw and to the 0.5 M salt buffer used to
prepare the P10 extracts. Protein concentrations in extracts were
determined using the Bio-Rad microprotein assay using bovine serum
albumin as standard.
Electrophoretic Mobility Shift Assays--
A synthetic
oligonucleotide corresponding to the consensus for p53 binding was as
described previously (40). DNA binding extracts contained 9 µg of
total protein in 25 mM Hepes, pH 7.5, 75 mM
KCl, 10% glycerol, 1 mM dithiothreitol plus 1 µg of
poly(dI-dC) as competitor DNA, 4 µl of the p53 monoclonal antibody,
Pab 421, and ~10 femtomoles of p53 oligonucleotide that had been
labeled at the 5' position with [32P]ATP (40). Samples
were set up on ice to contain buffer, competitor DNA, and protein, and
radiolabeled DNA was added last. Samples were incubated at room
temperature for 15 min then loaded directly on prerun, nondenaturing
polyacrylamide gels. Electrophoresis was as described previously
(41).
Western Blots--
SDS-polyacrylamide gel electrophoresis
and Western blots for DNA-PKcs and ATM were as described previously
(42). Chk2 and p53 were analyzed on 10% acrylamide SDS gels. A rabbit
polyclonal antibody to the rad3-like domain of ATM (4BA) was used as
described previously (35). Antibodies to p53 (Pab421 and DO-1) and p21 (Ab-4) were purchased from Oncogene Scientific. Phosphospecific antibodies to serine 15 of p53 and threonine 68 of Chk2 were as described previously (37, 43). A rabbit polyclonal antibody to Chk2 was
as previously described (32). Western blots were developed using ECL
(Amersham Pharmacia Biotech) according to the manufacturer's instructions.
Immunoprecipitation Assays--
ATM was immunoprecipitated from
cells as described by Canman et al. (34). Briefly,
~106 cells were pelleted and washed two times in
phosphate-buffered saline. Cells were lysed by sonication in
immunoprecipitation buffer (50 mM Tris-HCl, pH 7.5, 50 mM 
glycerol phosphate, 150 mM NaCl, 10%
glycerol, 1% Tween 20, 1 mM NaF, 1 mM
NaVO4, 1 mM phenylmethysulfonyl fluoride, 2 µg/ml pepstatin, 5 µg/ml leupeptin, 10 µg/ml aprotinin, and 1 mM dithiothreitol), and ATM was immunoprecipitated by a ATM
rabbit polyclonal antibody (Ab-3, Oncogene Scientific). Immunoprecipitates were washed two times with 500 µl of
immunoprecipitation buffer, twice with 100 mM Tris-HCl, pH
7.5, containing 0.5 M LiCl, followed by two washes in
pre-kinase buffer (10 mM Hepes, pH 7.5, 50 mM
-glycerophosphate, 50 mM NaCl, and 1 mM
dithiothreitol). ATM kinase activity was assayed in pre-kinase buffer
containing 10 mM MnCl2, 10 µM ATP
containing 5 µCi of [
-32P]ATP, and 0.5 µg of
protein substrate. Substrates used were PHAS-I (Stratagene), GST-tagged
recombinant Chk2 (amino acids 1-222), or recombinant GST-tagged p53
(amino acids 1-40) as described previously (35). Samples were analyzed
on 16% polyacrylamide SDS gels and exposed to Fuji x-ray film at
80 °C with intensifying screens for 12-16 h.
DNA Fragmentation Assays--
Genomic DNA was extracted from
~106 cells using DNAzol reagent (Life Technologies, Inc.)
exactly as described in the manufacturer's instructions. 20 µg of
DNA was run on 1.5% agarose submarine gels in Tris-borate-EDTA buffer
at 100 V for 1.5 h. Gels were stained with ethidium bromide for
1 h, and DNA was imaged under UV light using a BioGel scanner
(Bio-Rad).
Cell Survival Assays--
2 × 105 BT and L3
cells were cultured overnight in RPMI medium containing
penicillin/streptomycin and 10% fetal calf serum (Hyclone). Genistein
and etoposide were then added to the media to final concentrations of
0, 25, 50, 100, 200, and 300 µM and 6.8, 13.6, 34, 68, and 136 µM, respectively. After 2 h the drug was
removed, and the cells were washed twice in 1 ml RPMI/10% fetal calf
serum medium and allowed to grow for 5-7 days. Following incubation
with 5 µCi/ml tritiated thymidine (Amersham Pharmacia Biotech) for 90 min, cells were centrifuged at 1,500 × g for 5 min and
washed twice in phosphate-buffered saline. The cell pellet was lysed in
1 N NaOH and added to 1 ml of ReadySafe (Beckman) scintillation fluid for counting on the Beckman LS6500 scintillation counter.
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RESULTS |
Effects of Genistein on ATM-dependent
Signaling--
Exposure of cells to ionizing radiation results in
activation of ATM-dependent signaling pathways that leads
to activation of p53 and the protein kinase Chk2 (31). In the case of
p53, ionizing radiation results in an increase in total protein levels and phosphorylation of p53 at N-terminal sites, including serine 15. Cells that lack ATM show delayed up-regulation of p53 protein levels in
response to ionizing radiation. Consequently, phosphorylation of p53 at
serine 15, and DNA binding of p53 to its cognate DNA binding site is
reduced in A-T cells (reviewed in Ref. 30). In addition, ionizing
radiation results in ATM-dependent activation and
phosphorylation of Chk2, which in turn leads to phosphorylation and
inactivation of the dual specificity tyrosine phosphatase, cdc25
(32).
Genistein has been shown to induce stabilization of p53 protein levels
in normal cells but not in A-T cells (36), suggesting that it may
activate a pathway similar to that activated by ionizing radiation. To
further understand the mode of action of genistein, we first examined
whether genistein also induced phosphorylation of p53 at serine 15. A-T
cells and normal cells were exposed to genistein (100 µM)
for 2 h. Cells were then harvested, crude cytoplasmic (S10) and
nuclear (P10) extracts were prepared, and extracts were analyzed for
total p53 protein levels using DO-1 antibody. As shown previously,
genistein resulted in an increase in p53 protein levels in ATM-positive
cells, which were greatly reduced in A-T cells (Fig.
1A, upper panel).
When the same samples were probed with a phospho-specific antibody to
serine 15 of p53, p53 was seen to be highly phosphorylated in
genistein-treated control, but not A-T cells (Fig. 1A,
lower panel). We note that the reduced phosphorylation of
p53 observed in A-T cells may reflect lack of protein induction rather
than a decrease in the stoichiometry of p53 phosphorylation. The
effects of genistein on p53 are therefore highly similar to those
observed with ionizing radiation, in that both up-regulation of p53
protein and phosphorylation at serine 15 is greatly diminished in
ATM-defective (A-T) cells.

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Fig. 1.
Genistein activates p53 and Chk2 in
ATM-positive cells. A, Western blot of p53 levels and serine
15 phosphorylation in genistein-treated cells. Control lymphoblastoid
cells (BT, lanes 1-4) or A-T cells (L3, lanes
5-8) were incubated with genistein (100 µM,
lanes 3, 4, 7, and 8) or an
equal volume of Me2SO (lanes 1, 2,
5, and 6) for 2 h at 37 °C under
CO2 and humidity. S10 (S) and P10 (P)
extracts (cytoplasmic and nuclear respectively) were prepared in the
presence of phosphatase and protease inhibitors as described under
"Materials and Methods." 20 µg of total protein was analyzed by
Western blot using a panspecific antibody (DO-1) to p53 (upper
panel) or a phosphospecific antibody to serine 15 of p53
(lower panel). B, electrophoretic mobility shift
assay of p53 DNA binding activity. The same extracts were analyzed by
electrophoretic mobility shift assay for the ability of endogenous p53
to bind to DNA as described under "Materials and Methods."
ATM-positive cells (lanes 1-4) or ATM-negative cells
(lanes 5-8) were either treated with an equivalent volume
of Me2SO (lanes 1, 2, 5,
and 6) or genistein (100 µM, lanes
3, 4, 7, and 8) for 2 h.
Shown is an autoradiogram of an overnight exposure at 80 °C with
intensifying screens. C, Western blot of genistein treated
cells for p21 protein expression. ATM-positive (lanes 1,
2, 5, and 6) or ATM-negative
(lanes 3, 4, 7, and 8)
cells were exposed to 100 µM genistein for 2 h
(lanes 1-4) or irradiated with 10 Gy ionizing radiation and
allowed to recover for 2 h (lanes 5-8). 100 µg of
protein extract was analyzed by Western blot using a monoclonal
antibody to p21. IR, ionizing radiation.
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We next examined whether genistein affected the ability of p53 to bind
to a DNA oligonucleotide containing the p53 consensus sequence.
Treatment of ATM-positive cells with genistein was found to
dramatically increase the ability of p53 to bind to its specific DNA
sequence (Fig. 1B, lanes 1-4). Binding of p53 to
DNA was significantly reduced in A-T cells compared with normal cells
(Fig. 1B, lanes 5-8), again indicating that the
effect of genistein on p53 is very similar to that of ionizing radiation.
Activation of the sequence-specific DNA binding properties of p53
suggests that genistein might activate the transcriptional activation
activity of p53 and result in induction of downstream genes such as
p21. Western blotting with an antibody to p21 revealed that p21 protein
levels were elevated in response to genistein in ATM-positive cells
(Fig. 1C).
When ATM-positive cells are exposed to ionizing radiation, Chk2
undergoes a reduction in mobility on SDS-polyacrylamide gel electrophoresis/Western blot that has been shown to be the result of
phosphorylation (32). The same antibody was therefore used to probe for
changes in Chk2 mobility in response to genistein. Slower migration of
Chk2 was observed in control cells (ATM-positive cells) that had been
treated with genistein (Fig.
2A, lane 2) but was
completely absent in ATM-defective (A-T) cells (Fig. 2A, lane 6). The genistein-induced change in migration of Chk2
in control cells was indistinguishable from that observed by exposure to ionizing radiation (Fig. 2A, compare lanes 2 and 3).

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Fig. 2.
Western blot for Chk2 modification in normal
and A-T cell lines. A, a normal lymphoblastoid cell
line (BT, lanes 1-4) or an A-T line (L3, lanes
5-8) were treated with genistein (100 µM) for
2 h. S10 samples were prepared as described in the legend to Fig.
1A, and 20 µg of protein was analyzed by Western blot
using an antibody to Chk2. The phosphorylated form of Chk2 migrates
slightly slower than the unphosphorylated form of the protein, as
indicated by the arrows (32). Where indicated, cells were
irradiated at 10 Gy either in the absence (lanes 3 and
7) or the presence of genistein (lanes 4 and
8). The majority of the Chk2 was found in the S10 fraction
(shown), but similar results were observed in the P10 fraction (data
not shown). B, extracts were prepared exactly as in
A from either a normal cell line (C3ABR, lanes 1 and 2) or an A-T line containing mutant, inactive ATM
(AT1ABR, lanes 3 and 4) either in the absence
(lanes 1 and 3) or presence of genistein
(lanes 2 and 4). C, extracts were
prepared from BT or L3 cells (control and A-T, respectively) exactly as
in A. 100 µg of protein from the S10 fraction was loaded
onto a 10% SDS-polyacrylamide gel and blotted with an phosphospecific
antibody to threonine 68 of Chk2. IR, ionizing radiation.
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The cell lines used for these experiments were lymphoblastoid cell
lines from an A-T patient (L3) and a normal control (BT). This
therefore raises the possibility that the lack of effect of genistein
on Chk2 in the ATM-deficient cell line L3 could be due to some other
defect in these cells. We therefore repeated these experiments on
another pair of cell lines, C3ABR, which has normal levels of ATM, and
AT1ABR, which contains mutant inactive ATM. Again, genistein treatment
was found to result in modification of Chk2 only in the cell line
containing normal ATM (Fig. 2B, lanes 1 and
2). Similarly, phosphorylation of p53 at serine 15 was also
more pronounced in the normal cell compared with the A-T line (AT1ABR)
(data not shown). Together, these data strongly support the idea that
the effects of genistein on Chk2 are mediated through ATM.
It has recently been shown that ionizing radiation induces
ATM-dependent phosphorylation of Chk2 on threonine 68 (43, 44). We therefore used a phosphospecific antibody to threonine 68 (43) to examine phosphorylation of Chk2 in response to genistein. Exposure to genistein was found to induce phosphorylation of Chk2 at threonine 68 in ATM-positive cells but not in A-T cells (Fig. 2C). We
therefore conclude that ATM is essential for the phosphorylation of
Chk2 in response to exposure to both ionizing radiation and genistein.
Genistein Activates ATM Kinase Activity--
Exposure of cells to
ionizing radiation or the radiomimetic drug neocarzinostatin, results
in increased kinase activity of immunoprecipitated ATM (33, 34). We
therefore assayed for ATM kinase activity toward known in
vitro substrates, PHAS-I, Chk2, and p53 (33, 34, 35) in cells
treated with genistein. Treatment with genistein induced a modest but
reproducible increase in ATM kinase activity in ATM-positive cells
(Fig. 3) but not A-T cells (data not
shown), consistent with activation of a DNA damage response
pathway.

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Fig. 3.
ATM-positive (BT) cells were either untreated
(odd-numbered lanes) or treated with genistein
(100 µM) (even-numbered
lanes). After 2 h, extracts were made for
immunoprecipitation, and ATM kinase activity was assayed with protein
substrates at 0.5 µg. Substrates used were: recombinant GST-tagged
Chk2 (amino acids 1-222) (lanes 1 and 2), PHAS-1
(lanes 3 and 4), and recombinant GST tagged p53
(amino acids 1-40) (lanes 5 and 6).
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Caffeine and Wortmannin Abrogate the Effects of Genistein on p53
and Chk2--
The protein kinase activity of ATM and other
phosphatidylinositol 3-kinase-like proteins such as DNA-PKcs and
ATM-Rad3-related protein (ATR) is inhibited by treatment of
cells with wortmannin or caffeine (45-47). ATM-positive cells were
therefore treated with genistein plus either wortmannin or caffeine. As
shown in Fig. 4, wortmannin reduced
genistein-induced phosphorylation of p53 (on serine 15) and completely
abrogated the phosphorylation-dependent mobility shift of
Chk2 (lane 4). Caffeine, on the other hand completely abrogated both phosphorylation of p53 and the phosphorylation dependent mobility shift of Chk2 (Fig. 4, lane
6).

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Fig. 4.
Wortmannin and caffeine abrogate the
genistein induced activation of p53 and Chk2. Normal (BT) cells
were incubated with genistein (100 µM) (lanes
2, 4, and 6) in the presence of either 20 µM wortmannin (WM, lanes 3 and
4) or 10 mM caffeine (lanes 5 and
6) for 2 h. Extracts were prepared as previously and
assayed by Western blot using antibodies to either serine 15 of p53 or
Chk2.
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Genistein and the Topoisomerase II Inhibitor Etoposide, but Not the
Tyrosine Kinase Inhibitor Herbimycin A, Induce Phosphorylation of p53
and Chk2--
Genistein has been reported to act as both a broad
specificity inhibitor of protein-tyrosine kinases and an inhibitor of
topoisomerase II. To determine whether the effects of genistein on p53
and Chk2 were due to ATM-dependent inhibition of a tyrosine
kinase or of topoisomerase II, cells were treated with either
herbimycin A, a tyrosine kinase inhibitor that does not induce
apoptosis (48, 49), or etoposide. Like genistein, etoposide stabilizes
the covalent topoisomerase II-DNA cleavage intermediate, a reaction that is known to cause DNA breaks (11) and induce apoptosis (15)
in vivo. Etoposide induced phosphorylation of p53 on serine 15, a phosphorylation-dependent reduction in mobility of
Chk2, and phosphorylation of Chk2 on threonine 68 (Fig.
5A). In contrast, herbimycin
at concentrations known to inhibit tyrosine kinases (48-50) did not
induce significant phosphorylation of p53 or Chk2 (Fig. 5A).
From these data we conclude that genistein-induced activation of p53
and Chk2 is due to topoisomerase II induced damage rather than
inhibition of a tyrosine kinase.

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Fig. 5.
Etoposide but not herbimycin induces
phosphorylation of p53 and Chk2. A, control cells (BT) were
untreated (lanes 1 and 5), incubated with
etoposide (68 µM) (lanes 2-4), or herbimycin
A (lanes 6-8). At the times indicated, cells were
harvested, and 20 µg total protein was analyzed by Western blot using
antibodies to p53 (DO-1), serine 15 of p53, or Chk2 as indicated. In
the lower panel, 100 µg of protein was analyzed by Western
blot using a phosphospecific antibody to threonine 68 of Chk2.
B, control cells (BT, lanes 1-5) or A-T cells
(L3, lanes 6 and 7) were incubated with
herbimycin A at 1 µM (lane 1) or etoposide at
68 µM (lanes 2-7) for 0-24 h as indicated.
Extracts were made and blots were analyzed with antibodies to DO-1,
serine 15 of p53, and Chk2 as described for A.
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Our results clearly show that genistein activates p53 and Chk2 in an
ATM-dependent manner. Because genistein and etoposide both
act on topoisomerase II, we next asked whether, like genistein, the
action of etoposide is also ATM-dependent. Treatment of
cells with etoposide was found to induce phosphorylation of p53 at
serine 15, the phosphorylation-dependent mobility shift of
Chk2 (Fig. 5B), and phosphorylation of Chk2 on threonine 68 (data not shown). However, unlike genistein, none of these events was
dependent on the presence of ATM (Fig. 5B, lane
7). These data suggest that although genistein and etoposide both
act on topoisomerase II, they may act via different pathways, one that
requires ATM and one that does not.
Etoposide but Not Genistein Induces Protein and DNA
Fragmentation--
Among the hallmarks of apoptosis are
caspase-mediated cleavage of key protein substrates and fragmentation
of chromatin to produce characteristic DNA ladders. We and others have
shown that treatment of a variety of cell types with etoposide results
in cleavage of the ATM-related protein, DNA-PKcs, to produce an
N-terminal 240-kDa fragment and a 150-kDa C-terminal fragment (16-18).
Similarly, the ATM protein is cleaved during apoptosis to produce a
240-kDa fragment (19). We therefore treated ATM-positive (BT) and
ATM-deficient cells (L3) with genistein, etoposidem or the tyrosine
kinase inhibitor herbimycin A. Because etoposide induces cleavage of
both DNA-PKcs and ATM in HL60 cells, HL60 cells were used as a positive control.
Neither ATM nor DNA-PKcs proteins were cleaved when BT or L3 cells were
treated with genistein, or etoposide for 2, 4, 8, or 24 h (Fig.
6A and data not shown). Even
exposure of BT and L3 cells to 300 µM genistein for
24 h did not induce cleavage of DNA-PKcs or ATM (data not shown),
suggesting that genistein is not activating a caspase-mediated pathway
in these particular cells. Because etoposide also failed to induce
cleavage of ATM and DNA-PKcs, our data suggest that these cells may be
resistant to apoptosis. Treatment of HL60 cells with etoposide resulted in cleavage of both DNA-PKcs and ATM within 4 h of treatment (Fig. 6B); however, incubation with up to 300 µM
genistein had no effect on either protein in HL60 cells (data not
shown). Similarly, etoposide but not genistein induced DNA
fragmentation in HL60 cells (Fig. 6C). Neither etoposide nor
genistein induced cleavage of DNA in the BT or L3 cells under the
conditions used in this study (Fig. 6C).

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Fig. 6.
Etoposide but not genistein induces cleaved
of DNA-PKcs and ATM. A, control (BT) cells (lanes
1-4) or A-T cells (L3) (lanes 5-8) were incubated for
24 h with either etoposide (68 µM) (lanes
2 and 6), genistein (100 µM) (lanes
3 and 7), or herbimycin A (1 µM)
(lanes 4 and 8), or were untreated (lanes
1 and 5). Extracts were made as described for Fig.
5A and assayed by Western blot using an antibody (DPK1) to
amino acids 2018-2137 of DNA-PKcs or antibody 4BA to ATM as indicated.
B, HL60 cells were incubated with etoposide (68 µM) (lanes 1-4) or with an equivalent volume
of Me2SO (lane 5) for 0-8 h as indicated.
Samples were prepared for Western blot exactly as for Fig.
5A. C, total genomic DNA was extracted from cells
using DNAzol reagent (as described under "Materials and Methods"),
and 20 µg was analyzed on 1.5% agarose gels and stained with
ethidium bromide as described. Lane 1, HL60 cells,
untreated; lanes 2-4, HL60 cells treated with etoposide for
2, 4, or 8 h, respectively; lanes 5-8, ATM-positive
(BT) cells either untreated (lane 5) or incubated with
etoposide (lane 6), genistein (lane 7), or
herbimycin (lane 8) at 68, 100, or 1 µM,
respectively, for 24 h. Samples in lanes 9-12 were
derived from A-T cells (L3) that were treated with etoposide,
genistein, or herbimycin exactly as in lanes 5-8.
D, samples were prepared from etoposide treated HL60 cells
exactly as described for B. Samples were assayed for Chk2
phosphorylation using the antibody previously described (32).
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Incubation of HL60 cells (which do not contain p53 protein) with
etoposide also resulted in phosphorylation of Chk2 (Fig. 6D), indicating that etoposide induced activation of Chk2
can occur in the absence of an intact p53 damage response pathway.
Although our data suggest that genistein does not induce an apoptotic
pathway in BT and L3 cells, phosphorylation of p53 and Chk2 suggested
that a DNA damage response pathway was being activated. Because our
data clearly show that genistein preferentially acts on ATM-positive
cells, we reasoned that ATM-positive and -negative cells might have
different survival rates after exposure to genistein. Cells were
treated with either genistein or etoposide for 2 h, and cell
survival was determined as described under "Material and Methods."
As predicted, ATM negative cells were more susceptible to genistein
treatment than ATM-positive cells, particularly at higher doses (Fig.
7, left panel). In
contrast, etoposide induced similar levels of cell death in both cell
types at all doses used (Fig. 7, right panel).

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Fig. 7.
Cell survival analysis of BT and L3 cells
treated with genistein or etoposide. Cells were treated for 2 h with either genistein (left panel) or etoposide
(right panel) at the concentrations indicated. Cell survival
was measured as described under "Materials and Methods."
Squares indicate BT cells (ATM-positive, control cells), and
circles represent L3 cells (ATM-deficient cells). Results
represent the average of three separate experiments.
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DISCUSSION |
Genistein belongs to a family of naturally occurring
phytoestrogens that includes quercetin, biochanin A, kaempferol, and daidzein. These compounds have been reported to have a wide variety of
biological functions, including both growth promoting and
anti-tumorigenic effects. Genistein, in particular, has been reported
to have a plethora of biological activities, including inhibition of
tyrosine kinases, inhibition of topoisomerase II, anti-proliferative
effects in breast cancer cells, induction of growth arrest, and
apoptosis. Here we show that genistein induces
ATM-dependent up-regulation of p53, phosphorylation of p53
at serine 15, and activation of the sequence specific DNA binding
properties and transcriptional activation of p53. These and previous
data (36) suggest that genistein signals via ATM to p53 at the
G1/S cell cycle checkpoint.
Genistein also induced reduced mobility of Chk2 protein on Western
blot. The reduced mobility of Chk2 was indistinguishable from that
induced by exposure to ionizing radiation, an event that has previously
been shown to be due to phosphorylation. Moreover, we show that, like
ionizing radiation, genistein induces phosphorylation of Chk2 on
threonine 68. Significantly, genistein-induced phosphorylation of Chk2
was completely absent in two lymphoblastoid A-T cell lines, suggesting
that ATM is absolutely required for genistein-induced signaling through
Chk2. Also, our data show that cells containing ATM display increased
cell survival in the presence of genistein. We speculate that genistein
may activate an ATM-dependent DNA damage response pathway
that protects cells from induced DNA damage.
Recently, Darbon and colleagues (51) reported that genistein induced
phosphorylation of serine 15 of p53 and activation of Chk2 in a human
melanoma cell line and that the effects of genistein were abrogated by
caffeine, a known inhibitor of ATM, and the related protein, ATR
(45-47). In the study by Darbon et al. (51) ATM-deficient
cell lines were not examined; however, the authors concluded that ATM
was not involved in the genistein-induced activation of p53 and Chk2
because treatment of cells with wortmannin, a known inhibitor of
phosphatidylinositol 3-kinase-like protein kinases, including ATM, ATR,
and DNA-PK (24, 33-35), did not abrogate genistein-induced
phosphorylation of p53 and Chk2. In our study, wortmannin reduced
phosphorylation of serine 15 of p53 and completely abrogated
phosphorylation of Chk2. Moreover, we show that in two separate pairs
of lymphoblastoid cell lines, the effects of genistein on p53 and Chk2
are dependent on the presence of ATM. We speculate that these
differences may be due to the different types of cell lines used in the
two studies.
Although genistein is known to inhibit tyrosine kinases, the tyrosine
kinase inhibitor herbimycin A did not induce phosphorylation of Chk2 or
p53, suggesting that genistein is acting via a
topoisomerase-dependent DNA damage response pathway rather
than via inhibition of a tyrosine kinase. Genistein and etoposide have
both been reported to induce DNA damage via stabilization of the
topoisomerase II-DNA cleavage intermediate. Although both agents induce
phosphorylation of p53 and Chk2, our results show that only genistein
activates an ATM-dependent pathway and suggest that
etoposide is activating a different DNA damage response pathway than
genistein, that is not mediated by ATM. This suggests that other
protein kinases that phosphorylate p53 (at serine 15) and Chk2 at
threonine 68 and possibly other sites, must be activated by etoposide
treatment. Possible candidates include the related protein DNA-PK,
which phosphorylates threonine 68 of Chk2 and serine 15 of p53 in
vitro (Refs. 35 and 38 and data not shown) and ATR, which
phosphorylates serine 15 of p53 and threonine 68 of Chk2 in
vitro (44, 50) and plays a role in the ionizing radiation-induced
stabilization of p53 in vivo (50-53).
Although genistein clearly activates a DNA damage response pathway, the
type of damage induced by genistein remains unclear. Genistein did not
induce DNA fragmentation in BT, L3, or HL60 cell lines used in this
study. Similarly, genistein was only weakly effective in causing DNA
damage in melanoma cells, as measured by the single cell alkaline
electrophoresis (Comet) assay (51). However, these results do not
preclude the possibility that genistein induces a subtle form of DNA
damage that was not efficiently detected by either assay method.
Although genistein and etoposide both inhibit topoisomerase II,
in vitro studies indicate that genistein and etoposide
induce different types of DNA damage and that genistein but not
etoposide also induces protein-DNA cross-links (9). It is therefore
conceivable that ATM is required for repair of some types of DNA
damage, breaks, or cross-links but not others. Another possibility is
that the combined effects of genistein on tyrosine kinases and
topoisomerase II selects for or causes a specific type of lesion, the
repair of which requires ATM. Interestingly, the protein-tyrosine
kinase, c-Abl, has been shown to interact with ATM and to play a role in signaling of DNA damage (54, 55) and, therefore, could play a role
in the cellular response to genistein.