[CANCER RESEARCH 63, 2891–2897, June 1, 2003]
Histone Deacetylase Inhibitors Activate p21WAF1 Expression via ATM
Rong Ju, and Mark T. Muller1
Department of Molecular Genetics, The Ohio State University, Columbus, Ohio 43210
ABSTRACT
Histone deacetylase (HDAC) inhibitors are known to induce expression
of genes such as p21WAF1, thereby, leading to cell cycle arrest. In this work,
we show that p21WAF1 induction by HDAC inhibitors (depsipeptide and
trichostatin A) is defective in Ataxia telangiectasia (AT) cells but normal
in matched wild-type (WT) cells (human diploid fibroblasts). To verify the
role of ATM in this effect, we show that ectopic expression of the WT
ATM gene in an AT cell line fully restores p21WAF1 induction by the
HDAC inhibitors. Furthermore, because caffeine and wortmannin attenuate p21WAF1 induction in WT cells, it is probable that the phosphatidylinositol 3ⴕ-kinase activity is essential for this process. Besides the p21WAF1
promoter, activation of topoisomerase III␣ and SV40 promoters by the
HDAC inhibitors are also decreased in the AT cell lines relative to WT
cells; thus, these findings pertain to other promoters. Finally, despite the
obvious induction deficiency of gene expression, the overall levels of H3
and H4 histone acetylation appear to be the same between AT and normal
cells in response to HDAC inhibitor treatments. Taken together, the data
indicate that ATM is involved in histone acetylation-mediated gene regulation.
INTRODUCTION
In eukaryotic cells, DNA combines with core histones and other
chromosomal proteins to form chromatin (1, 2) where two copies of
each of four inner histones are wrapped by 146 bp of DNA to make
a nucleosome, the basic repeating unit. With the aid of additional
proteins, including histone H1, nucleosomes are additionally organized into higher-order chromatin structures (3). The presence of
histones on DNA limits access of nonhistone DNA-binding proteins
and thereby represses transcription (4). To date, at least two distinct
mechanisms are thought to alter the repressive nature of bulk chromatin vis a vis gene expression (5–7). One mechanism involves
ATP-dependent nucleosome remodeling by which chromatin remodeling complexes transfer and slide histone cores, making DNA accessible to transcription factors and DNA binding factors that promote
transcription (6, 8, 9). The second mechanism involves factors that
directly modify core histone tails through phosphorylation, methylation, and acetylation (10, 11). Acetylation-related factors include
HATs2 and HDACs (12–14), which acetylate and deacetylate, respectively, lysine residues at the NH2-terminal tails of the histone core
particle (15). Because the histone tails are located outside of the
particles, such modifications are believed to neutralize the positive
charge of basic histones and weaken histone-DNA interactions, thus
making the nucleosome more “accessible.” Consequently, HATs activate transcription (16, 17), whereas HDACs remove acetyl groups,
leading to a more repressive form of chromatin (18). The dynamic
equilibrium of HATs and HDACs determines the net level of acetyReceived 7/22/02; accepted 4/2/03.
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1
To whom requests for reprints should be addressed, at Department of Molecular
Genetics, Ohio State University, 484 West 12th Avenue, Columbus, OH 43210. Phone:
(614) 292-1914; Fax: (614) 292-4702; E-mail: [email protected].
2
The abbreviations used are: HAT, histone acetyltransferase; HDAC, histone deacetylase; AT, ataxia telangiectasia; ATM, ataxia telangiectasia-mutated; PI3k, phosphatidylinositol 3⬘-kinase; ATR, ataxia telangiectasia-mutated-related protein kinase; Rb, retinoblastoma; TSA, Trichotatin A; topo, topoisomerase; PMSF, phenylmethylsulfonyl
fluoride; TBST, 0.2 mM Tris-HCl (pH 7.5), 150 mM NaCl, 0.1% Tween; FR, depsipeptide;
WT, wild-type; pol II, polymerase II.
lation and, in turn, regulate transcription. At present, several classes of
HATs (12–14) and HDACs (19 –22) have been identified in yeast and
human cells. In general, transcriptional coactivators recruit HATs to
promoter regions to activate the corresponding genes (23, 24),
whereas transcriptional corepressors recruit HDACs to shut down
expression of the genes (25–27); thus, both function in complexes (13,
28). However, the exact mechanism of how histone acetylation activates transcription remains unclear.
AT is an autosomal recessive disorder in which patients exhibit
cerebellar ataxia, dilated blood vessels, immunodeficiency, hypersensitivity to ionizing radiation, and elevated risk of certain cancers (29,
30). Cells derived from AT patients display several cellular defects
including chromosome instability (31), defective cell cycle checkpoints (G1, S, and G2; Refs. 32, 33), and radioresistant DNA synthesis
(34). The mechanistic reason is that these cells fail to activate damage
response signaling, such as p53-dependent pathway (35). In addition,
a study has revealed that a significant decondensation of the nuclear
chromatin was associated with the AT disorder (36, 37).
The ATM gene was identified by positional cloning and encodes a
3056-amino acid protein with a calculated molecular weight of Mr
350,000. ATM shares homology with a gene family in which all of the
members have a COOH-terminal 300 amino acid motif that resembles
the catalytic domain of PI3k. Members of this gene family are
typically involved in DNA damage detection and cell cycle control
(38 – 40); examples include Saccharomyces cerevisiae proteins TEL1
and MEC1 (41, 42), Schizosaccharomyces pombe RAD3 (43),
Drosophila MEI-41 (44), ATR (45), and human DNA-pyruvate
kinase (46). The cumulative data indicate that the ATM gene product
is a protein kinase rather than a lipid kinase (38, 47). Consistent with
the central roles of ATM protein in cell cycle control and DNA repair,
it has multiple substrates including p53, Chk2, p95/NBS, BRAC1, and
MDM2 (48, 49). Although it is reported that ATM phosphorylates
HDAC1 (50) and ATR is associated with HDAC2 (51), there have not
been any reports that functionally link ATM to histone acetylationmediated gene expression.
p21WAF1 is a cyclin-dependent kinase inhibitor that associates with
a class of cyclin-dependent kinases and inhibits their kinase activities
leading to cell cycle arrest and dephosphorylation of Rb (reviewed in
Ref. 52). HDAC inhibitors have been reported to consistently induce
p21WAF1 expression in a p53-independent manner (53). In this study,
we used p21WAF1 as a model system to reveal whether ATM is
required for histone acetylation-mediated gene activation. We show
that such activation is defective in AT cells and that the PI3k activity
appears to be necessary to HDAC inhibitor-induced p21WAF1 activation in WT cells.
MATERIALS AND METHODS
Cell Cultures. Cells were grown in a humidified incubator at 37°C and 5%
CO2. GM 09607, GM05849, GM08505, GM00639, and GM00637 were purchased from Coriell Cell Repository (Camden, NJ). They were grown as
monolayers in DMEM (Life Technologies, Inc.) containing 15% fetal bovine
serum (Life Technologies, Inc.), 2⫻ concentrations of essential amino acid,
and 50 ␮g/ml of gentamicin. AT22IJE pEBS7 and AT22IJE pEBS7-YZ5 cells
were generous gifts from Dr. Michael B. Kastan at St. Jude Children’s
Research Hospital (Memphis, TN). They were originally constructed in the
laboratory of Dr. Yosef Shiloh (Tel Aviv University, Tel Aviv, Israel) by
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transfecting an immortalized fibroblast line AT22IJE with the mammalian
expression vector pEBS7 and pEBS7 plus full-length ATM reading frame,
respectively (54). AT22IJE pEBS7 and AT22IJE pFBS-YZ5 cells were cultured in the same medium as described above plus 100 ␮g/ml hygromycin B.
All of the cells were in exponential growth phase at the time of harvest.
Drugs and Antibodies. TSA was purchased from Sigma (St. Louis, MO)
and dissolved in DMSO at 100 mg/ml. FR 901228 was a kind gift from
Fujisawa Pharmaceutical Co., Ltd. (Osaka, Japan) and dissolved in DMSO at
5 mM. Aliquots were stored at ⫺20°C until immediately before the experiments.
Mouse monoclonal p21WAF1 antibody was from PharMingen (Los
Angeles, CA). Actin antibody was purchased from Sigma. Rabbit antiacetylated histone H3 and H4 antibodies were from Upstate Biotechnology
(Lake Placid, NY) and mouse anti-Rb antibody was purchased from Calbiochem (Los Angeles, CA).
Isolation of the p21WAF1 and the Topoisomerase III␣ Promoters. To
amplify the p21WAF1 promoter, two primers were designed and synthesized
according to Richon et al. (55). The sequences of upstream and downstream
primers were: 5⬘-GGT GTC TAG GTG CTC CAG GT-3⬘ and 5⬘-CCG GCT
CCA CAA GGA ACT GA-3⬘, respectively. The PCR reaction was carried out
in the following condition: 50°C 45 s, 72°C 30 s, and 94°C 1 min and 30
cycles. The PCR fragment was subcloned into pCRII-TOPO vector (Invitrogen, Carlsbad, CA) to yield pCRII-p21p. All of the orientations were examined
by sequencing.
The 659-bp KpnI-XhoI fragment of the p21waf1 promoter was cut out of
pCRII-p21p and cloned into pGL3 vector (Promega, Madison, WI) and designated
p21-luc. To amplify the topo III␣ promoter, two primers were synthesized as
following according to the published topo III␣ promoter sequence (56): upstream
5⬘-ATA GGT ACC CAA AAC GGC CTC ACG AAG CCA C-3⬘ and downstream 5⬘-TCA CTC GAG TCT TCG GGC CGT CGC AGC CAC CGG A-3⬘.
This fragment spanned from ⫹305 to ⫺1, 262 covering YY1, USF, and four Sp1
sites. Genomic DNA was extracted from GM0637 and used as the template. The
PCR reaction was carried out in the following conditions: 94°C 1 min, 72°C 1 min,
and 60°C 1 min and 30 cycles. Next, the 1.5-kb PCR fragment was digested with
KpnI and XhoI and subcloned into Bluescript II SK-phagemid to create pBStopoIIIp. After the topo III␣ promoter fragment was sequenced, pBS-topo IIIp was
digested with KpnI and XhoI, and the 1.5-kb fragment of topo III␣ promoter was
subcloned into pGL3 to yield pTopoIII-Luc.
Transient and Stable Transfection as well as Luciferase Analysis. For
transient assay, 1 ⫻ 105 GM0637 or GM5849 cells were transfected with
p21-luc or pTopoIII-luc (0.5 ␮g/transfection) using LF2000 (2 ␮l/␮g DNA;
Life Technologies, Inc.) in 24-well plates. After 24 h, transfected cells were
trypsinized/subcultured, and 24 h later the medium was replaced with either
fresh medium containing HDAC inhibitors or 0.1% DMSO. After an additional
24 h, cells were lysed and assayed for luciferase using a commercial kit
(Promega). For stable transfection, 1 ⫻ 105 GM0637 or GM5849 cells were
cotransfected with linearized pTopoIII␣-Luc or p21-luc (0.5 ␮g/transfection)
plus pcDNA3.1 (0.05 ␮g/transfection; Invitrogen), and 24 h later, the cells
were trypsinized and split into two 100-mm Petri dishes at a ratio of 1:9 to give
an appropriate number of colonies. After another 24 h, the G418 was added
into the medium to a final concentration of 800 ␮g/ml, and 2 weeks later, the
G418-resistant colonies were transferred to the new plates and amplified. For
the HDAC inhibitor induction assay, equal numbers of cells from each individual clone or pooled clones were loaded into two 24-well microtiter plates,
and 24 h later the old medium was replaced with medium containing HDAC
inhibitor or 0.1% DMSO. The cells were then lysed and assayed for luciferase
activity 24 h later.
Histone Extractions and Analysis. Histones were extracted by using a
modified procedure from Yoshida and Beppu (57). Briefly, cells were pelleted,
washed with PBS, and then suspended in buffer A [100 mM NaCl, 50 mM KCl,
20 mM Tris-HCl (pH 7.5), 0.1 mM EDTA, 1 mM PMSF, 10% glycerol, 0.2%
NP40, and 0.1% Triton X-100] to release nuclei. The nuclei were deposited by
centrifugation (1000 ⫻ g for 5 min; 4°C), the cytosol fraction removed and
nuclei resuspended in 100 ␮l of ddH2O. Concentrated H2SO4 (1 ␮l) was then
added and the extract incubated on ice for 1 h to dissolve the acid-soluble
nuclear proteins. The acid-insoluble fraction was removed by centrifugation
(14,000 ⫻ g 10 min). The acid-soluble proteins were precipitated with 1 ml of
acetone (4°C overnight). Precipitates were recovered by centrifugation at
12,000 ⫻ g for 10 min at 4°C. The pellet was suspended in solubilizing buffer
[0.1% SDS, 100 mM Tris-HCl (pH 6.8), 1 mM PMSF, and 1 ␮g/ml aprotinin,
leupeptin, pepstatin each], and the insoluble fraction was removed by centrifugation at 12,000 ⫻ g for 10 min. The protein concentration of supernatant was
determined using a Bio-Rad DC protein assay kit according to the manufacturer instructions (Bio-Rad, Hercules, CA).
Western Blot Analysis. To obtain a whole cell extract, cells were pelleted,
washed with PBS, and then suspended in radioimmunoprecipitation assay
buffer [50 mM Tris-HCl (pH 7.4), 1% NP40, 0.25% sodium deoxycholate,
1 mM EDTA, 1 mM PMSF, 1 ␮g/ml aprotinin, leupeptin, and pepstatin each,
1 mM Na3VO4, and 1 mM NaF] on ice for 10 min. The cell debris was removed
by centrifugation at 12,000 ⫻ g for 10 min. Protein concentration of the
supernatant was determined using a Bio-Rad DC protein assay (Bio-Rad).
Proteins were loaded onto SDS-polyacrylamide gels and transferred to Hybond
enhanced chemiluminescence membranes (Amersham Pharmacia Biotech,
Inc., Piscataway, NJ). Membranes were blocked for 2 h in TBST containing
5% nonfat dried milk, probed overnight with primary antibodies in TBST,
followed by a 2-h incubation in the secondary antibody in TBST, and visualized using BM Chemiluminescence’s Western Blotting kit according to the
manufacturer’s instructions (Roche Diagnostics, Indianapolis, IN).
RNA Isolation and Northern Blot Analysis. Total RNA was prepared by
using RNeasy Mini kit (Qiagen, Valencia, CA) according to the manufacturer’s instructions. Northern blotting was performed as described by Sambrook
et al. (58) with modifications. Briefly, total RNA (5 ␮g) was denatured in
formaldehyde and formamide buffer, fractionated on 1.2% formaldehyde agarose gel, and transferred overnight to Hybond-N⫹ nucleic acid transfer membranes (Amersham Pharmacia Biotech Inc., Piscataway, NJ). A fragment of
p21WAF1 cDNA only containing the coding sequence was isolated by digesting
the plasmid pCEP-WAF1 (Dr. Bert Vogelstein, the Johns Hopkins University,
Baltimore, MD) with HindIII and EcoRI, and was labeled with [␣32P]dCTP by
using the random-primed DNA labeling kit according to the manufacturer’s
instruction (Boehringer Mannheim, Indianapolis, IN). Prehybridization was
carried out by incubating blots in ExpressHyb Hybridization Solution (Clontech, Palo Alto, CA) for 2 h at 65°C. At the end of prehybridization, the
denatured radioactive probes were directly added into the solution to make the
final radioactivity at 106 cpm/ml, and hybridizations were conduct at 65°C
overnight. Blots were washed with 1⫻ SSC, 0.1% SDS once at the room
temperature, followed by three washes at 65°C with 0.2⫻ SSC, 0.1% SDS
before exposed to X-ray film for 48 h at ⫺70°C.
RESULTS
Induction of p21WAF1 by HDAC Inhibitors Is Defective in AT
Cells. FR is a potent HDAC inhibitor (59), and like other HDAC
inhibitors, FR induces p21WAF1 in a p53-independent manner in WT
cells (60). To test whether FR can induce p21WAF1 expression in AT
cells, SV40-transformed AT cells (GM5849) were treated with FR at
different concentrations and for different intervals. As a control, a
matched, SV40-transformed WT cells (GM0637) were treated in the
same way. Fig. 1A reveals that in GM637, p21WAF1 was induced by
FR at as low as 5 nM and saturated at 50 nM. In contrast, in the AT cell
line (GM5849), p21WAF1 was barely observed even when the cells
were treated with FR at a concentration as high as 500 nM. Furthermore, whereas the GM0637 cells started expressing p21WAF1 within
12 h after addition of FR (and reached a significant high level at 24 h),
p21WAF1 was hardly detected at 24 h in the GM5849 cells (Fig. 1B).
In both experiments, the ␤-actin levels that served as a loading control
stayed constant. These data indicate that FR cannot effectively induce
p21WAF1 expression in AT cells. On the basis of these results, we
performed subsequent experiments by using FR (50 nM) for 24 h
except where indicated.
To test whether such an induction deficiency is specifically because
of HDAC inhibition of FR, another structurally unrelated HDAC
inhibitor, TSA, was used to treat both cell lines. Similar results were
obtained as shown in Fig. 1C.
To assess whether p21 induction deficiency only occurs in GM5849
and to exclude the possibility that secondary mutations might be
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Fig. 1. Defective p21WAF1 induction by HDAC
inhibitors in AT cells. WT (GM0637) or AT
(GM5849) cells were cultured with the indicated
concentrations, times of exposures, and specific
HDAC inhibitor. A, cells were treated with 0 –500
nM FR for 24 h; B, cells were treated with 50 nM FR
for the indicated times; C, cells were incubated
with 0.5 ␮g TSA/ml for 24 h; D, WT (GM0639) or
AT (GM9607) cells were treated with FR (50 nM)
or TSA (0.5 ␮g/ml) for 24 h; E, various cell lines
were tested for FR induction (50 nM for 24 h):
GM0637 (WT), GM8505 (Bloom’s syndrome),
GM0639 (galactosemia cell line), and GM5849
(AT cell line). Cells were lysed and each extract
(100 ␮g) was loaded onto a 15% SDS polyacrylamide gel. P21WAF1 was detected by using a
mouse monoclonal anti-p21WAF1 antibody probed
as described in “Materials and Methods.” A parallel
blot was carried out to detect actin with a corresponding mouse antibody to serve as a loading
control. F, Northern blot analysis of p21WAF1
mRNA expression. Total RNA (10 ␮g) was isolated and fractionated on 1.2% agarose, and transferred to a nylon membrane. The membrane was
probed with 32P random-labeled human p21WAF1
cDNA. A parallel gel was stained with ethidium
bromide for a loading control.
responsible (because of an unstable genome caused by loss of ATM),
another SV40-transformed AT cell line GM9607 was also incubated
with both FR and TSA. Although p21WAF1 induction deficiency in
GM9607 (50% induction compared with WT) is not as severe as in
GM5849 (10% induction compared with wild type), p21WAF1 induction by both FR and TSA indeed decreased significantly when compared with the WT cells (Fig. 1D). These results suggest that it is
unlikely that the impaired p21WAF1 expression is because of the
ATM-unrelated secondary mutations. In addition, to confirm that our
results were specific to AT cells, SV40-transformed Bloom’s syndrome cell line GM8505 and galactosemia cell line GM0639 were
treated with FR. Galactosemia is an autosomal recessive disorder with
the deficiency of galactose-1-phosphate uridyltransferase (61).
Fig. 1E shows that both of these cell lines expressed similar levels of
p21WAF1 to the WT cells in response to FR; therefore, p21WAF1
induction deficiency appears to be AT-specific. Finally, to determine
whether the induction deficiency occurred at protein levels or mRNA
levels, p21WAF1. mRNA from both GM0637 (WT) and GM5849 (AT)
cells treated with various concentrations of FR was examined via
Northern blot analysis. Fig. 1F indicates that p21WAF1 m RNA levels
markedly increased in GM637 cells after cultured with FR at as low
as 5 nM and reached the saturated level at 50 nM. On the other hand,
although the level of p21WAF1 mRNA in GM5849 elevated in a
dose-dependent manner after FR treatments, the degree of induction is
only minor compared with the one in GM5849.
HDAC Inhibitor-mediated Induction Deficiency Occurs at Promoter Levels and Is Not Confined to p21WAF1. A 650-bp promoter
fragment for p21WAF1 was amplified by PCR using DNA from WT
cells (GM0637). The PCR product was validated by sequencing (data
not shown). To examine whether HDAC inhibitor-mediated induction
deficiency was transcriptional or post-transcriptional, the p21WAF1
promoter was fused with the luciferase gene, and the construct was
transfected into both the AT and WT cell lines. Fig. 2A shows that in
WT GM0637 cells, with increased concentration of FR, the episomal
p21WAF1 promoter was activated from 38-fold (5 nM FR) to as much
as 80-fold (500 nM FR). In contrast, there was an across the board
20-fold induction of the promoter activity in AT cells that did not
respond to increases in FR concentration. A similar profile was
observed when the p21WAF1 promoter was integrated into genome,
except the differences between the WT and AT cell was more dramatic (Fig. 2C). In AT cells, the integrated p21WAF1 promoter was
activated only ⬃5-fold on average in response to FR of all three
concentrations, compared with 20-fold as unintegrated plasmid in
transient assays. On the other hand, in the WT cells, both the integrated and episomal p21WAF1 promoter displayed a high level of
activation (80-fold by 50 nM of FR). Different from the results of
transient assays, the integrated p21WAF1 promoter appeared to be
saturated at 50 nM of FR. Thus, the results from stable assays appear
to be more consistent with Western blot data shown in Fig. 1. Taken
together, these data indicate that the p21WAF1 induction deficiency is
because of poor p21WAF1 promoter activation.
HDAC inhibitors affect expression of about 1–2% of all of the
genes (62); therefore, it was of interest to see whether loss of ATM
affected other genes besides p21WAF1. As shown in Fig. 2B, the
topoIII␣ promoter appears to be activated up to 6-fold in WT cell line
GM0637, whereas in the AT cell line GM5849, little if any activation
was seen. A constitutive promoter from SV40 also revealed similar
activation patterns. In addition, after stable transfection of topo III␣
promoter-luciferase gene into normal and AT cell lines, the integrated
topo III␣ promoter was activated significantly higher in normal transfectants relative to AT clones in response to FR (Fig. 2D). It is also
worth noting that in normal cells, the topo III␣ promoter was activated
at different degrees in the various transfected clones.
Ectopic Expression of ATM Restores the Defective p21WAF1
Induction by FR901228 in AT Cells. Genomic instability is a hallmark of ATM deficiency (31) largely because of random secondary
mutations. If HDAC inhibitor-mediated p21WAF1 induction deficiency in AT cells is because of these types of mutations, it should be
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Fig. 2. Promoter activation by FR in WT and AT cells. WT (GM0637) and AT (GM5849) cells were transfected in transient or stable assays, then transfected cells were treated
with or without FR, and promoter activation was analyzed. A, cells of both lines were transfected with p21-luc in transient assay, and after transfection, cells were cultured with 0.1%
DMSO (⫺FR) or with indicated concentrations of FR (⫹FR) for 24 h; B, cells of both lines were transfected with in transient assay, and transfected cells were treated with or without
FR (50 nM) for 24 h; C, cells of both lines were transfected with p21-luc in stable assay and G418-resistant clones (⬎100) from each cell line were pooled and treated with or without
FR (50 nM); D, cells of both cell lines were stably transfected with pTopo III-luc and individual G418-resistant clones (6 from each cell line) were treated with or without FR (50 nM).
After FR treatment, cells were lysed, and luciferase activity was measured. Mean fold increase from triplicate samples was determined by normalizing FR-treated luciferase to
DMSO-treated luciferase activity. The experiment was repeated at least three times, and the typical one was presented; bars, ⫾SD.
irreversible. That is, ectopic expression of a WT ATM in AT cells
should not restore the defective induction. To test this, we obtained
two AT fibroblast lines, AT22IJE pEBS7, derived from an immortalized fibroblast line AT22IJE transfected with an “empty” mammalian
expression vector pEBS7, and AT22IJE pFBS-YZ5, derived from the
same cell lines but transfected with an ATM cDNA (54). As shown in
Fig. 3A, AT cells expressed barely detectable levels of p21WAF1 after
FR treatment. On the other hand, p21WAF1 expression in ATMcomplemented cells was slightly less than in the WT cell line
Fig. 3. Ectopic expression of ATM restores p21WAF1 induction by FR in AT cells. An
isogenic pair of human AT fibroblast lines cell lines differing only in their ATM status,
AT22IJE pEBS7 and AT22IJE pFBS-YZ5, as well as a WT (GM0637) cell line were
treated with or without 50 nM FR (A) or 500 ng/ml TSA (B) for 24 h. Cells were harvested
and cell extract (100 ␮g) was loaded onto a 15% SDS polyacrylamide gel. p21WAF1 was
detected by using an anti-p21WAF1 antibody probed as described in “Materials and
Methods.” A parallel blot was carried out to detect actin to serve as a loading control.
GM0637. The similar result was seen after FR was replaced with TSA
(Fig. 3B). The data suggest that expression of the WT ATM gene
restores the p21WAF1 induction deficiency in AT cells and that the
lack of HDAC inhibitor induction appears to be a direct consequence
of the loss ATM activity, as opposed to some downstream secondary
mutations.
PI3k Activity and Induction of p21WAF1 by HDAC Inhibitors.
PI3k activity of ATM appears to be closely connected to many
cellular defects in AT cells (63). For example, expression of a truncated ATM gene lacking the PI3k domain confers the AT phenotype
on WT cells (63). To test the role of PI3k activity on ATM-mediated
p21WAF1 expression, the PI3k inhibitors caffeine and wortmannin
were used to treat the WT cells (GM0637) with FR and TSA. As
shown in Fig. 4A, combinations of either caffeine with FR or wortmannin with FR induced less p21WAF1 relative to FR alone. A similar
result was obtained with TSA (Fig. 4B). Moreover, caffeine inhibited
FR-mediated p21WAF1 promoter activation (Fig. 4C). Using the
pooled clones carrying integrated the p21WAF1 promoter, FR alone
induced the p21WAF1 promoter activity by ⬎60-fold at 5 nM and
80-fold at 50 nM FR or higher concentration, whereas addition of
caffeine to FR activated the p21WAF1 promoter by only 30-fold at the
same concentrations of FR. These results suggest that caffeine and
wortmannin repress the FR-mediated p21WAF1 induction at least
partially at the transcriptional level.
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(65). The similar results were seen in another AT cell line GM9607
(data not shown).
H3 and H4 Histone Acetylation in WT and AT Cells. To determine whether defective promoter activation was because of alterations
in histone acetylation, acid-soluble nuclear proteins were isolated
from WT and AT cells after exposure to FR for 0, 2, 4, and 8 h.
Western blot data (Fig. 6) show that before incubation with FR (0 h),
the levels of H3 and H4 were low in both the normal and AT cells
(Fig. 6). After FR addition, acetylated H3 and H4 started to accumulate equally in both cell lines. There was a relatively minor difference
in H4 acetylation kinetics in AT cells (see acetylated H4 in AT blot,
Fig. 6); however, other differences were not obvious between the two
cell lines.
DISCUSSION
Fig. 4. Effects of caffeine and wortmannin on p21WAF1 induction by FR and TSA. WT
(GM0637) cells were cultured with HDAC inhibitors alone or combination of HDAC
inhibitors and PI3k inhibitors for 24 h. A, caffeine (2 mM) or wortmannin (10 ␮M) was
combined with or without FR to treat WT cells; B, caffeine (2 mM) was combined with
TSA (0.2 ␮g/ml) to treat WT cells. In both assays, p21WAF1 protein was detected by
Western blot as described in Fig. 1; C, caffeine was combined with or without FR to treat
cells from the pooled GM0637 clones carrying p21WAF1-luc gene. Luciferase activity was
analyzed to determine fold increase as described in Fig. 2; bars, ⫾SD.
Fig. 5. Effect of FR on phosphorylation of Rb in AT cells. AT cell line (GM5849) and
a WT (GM637) cell line were treated with or without FR 50 (nM) for 24 h. Cells were
harvested to prepare the crude protein extract. Extract (100 ␮g) was loaded onto a 8% SDS
polyacrylamide gel. Rb was detected with an anti-Rb antibody probed as described in
“Materials and Methods.”
FR Fails to Induce Rb Dephosphorylation in AT Cells. Dephosphorylation of Rb is one downstream consequence of p21WAF1 in the
G1 checkpoint control pathway (reviewed in Ref. 64). To examine the
effect of FR on Rb phosphorylation status in AT cells, we carried out
Western blotting by using the antibodies against Rb. As shown in Fig.
5, elevated p21WAF1 is correlated with decreased Rb phosphorylation
in WT cell line GM637 in the presence of FR; however, in the AT cell
lines, GM5849, the levels of hyperphosphorylated Rb remained nearly
unchanged in response to FR. Moreover, the AT cells displayed
higher levels of Rb than the WT cells, consistent with previous studies
Previous studies revealed that both FR and TSA induce the expression of p21WAF1 in ATM WT cells (60, 66, 67). In this study, we
found that p21WAF1 induction by HDAC inhibitors (FR and TSA) was
defective in the AT cells. Because the defect was observed in three
independent AT cell lines and not in the other non-AT mutant lines
such as Bloom’s syndrome and galactosemia, it is unlikely that this
particular defect is because of random mutations unrelated to loss of
ATM gene function. Among these AT cell lines, the degree of the
defects appears different (Fig. 1), suggesting that different types of
ATM mutations may have distinct effects.
Like the other HDAC inhibitors, FR and TSA affect many biological processes, such as differentiation and apoptosis of transformed
cells in culture and inhibition of tumor growth (60, 68 –70). The
global effects of FR and TSA may reflect the multiple roles of
HDACs in chromatin. It is also possible that FR or TSA affects other
physiological targets and processes besides HDACs; however, given
their complete unrelated structures, any common effects caused by
both TSA and FR are likely to be because of an HDAC target.
Therefore, we conclude that defective p21WAF1 induction by FR or
TSA is associated directly with the expected target for these drugs,
HDACs.
The fact that ectopic expression of the ATM gene restores defective
p21WAF1 induction supports the notion that ATM is associated directly with HDAC inhibitor-mediated p21WAF1 induction. The data
also suggest that repression of FR/TSA-induced p21WAF1 expression
by caffeine or wortmannin is most likely related to ATM (instead of
ATR), although ATR has been reported to associate with HDAC2
(51). Taken together, we conclude that a functional ATM is essential
for histone acetylation-dependent p21WAF1 expression. Whereas both
caffeine and wortmannin behaved similarly, the former drug was a
more efficient inhibitor of the FR/TSA-mediated p21WAF1 induction
for reasons that are not clear (note that we cannot rule out stability
Fig. 6. Effect of FR on histone acetylation in AT cells. WT (GM0637) and AT
(GM5849) cells were treated with 50 nM FR for the indicated times. Nuclei were isolated,
and histones were acid extracted. A total of 5 ␮g histones were loaded onto a 20% SDS
polyacrylamide gel. Acetylation was detected by using antiacetylated H3 and H4 antibodies, respectively. A parallel gel was stained with Coomassie blue as a loading control.
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differences between caffeine and wortmannin over the 24-h incubation in vitro).
Although FR and TSA inhibit most HDACs, they only affect
expression of ⬃2% of mammalian genes (62). Therefore, it was of
interest to determine the generality of the response with other promoters besides p21WAF1. To address this, we tested two other promoters, SV40 and topoIII␣. The topoIII␣ promoter is TATA-less,
sharing similarity to a number of housekeeping genes (56). The SV40
promoter is a constitutive “promiscuous” promoter with the prototypic
TATA box and has shown to be activated in normal cells by TSA and
FR (59). Despite these differences, both promoters appear to be
induced by the HDAC inhibitors in WT human cells in this study.
However, activation was clearly diminished in AT cells. From these
data we conclude that the defect in acetylation-dependent gene expression in AT cells is not limited to p21WAF1 alone.
In stably transfected WT cells containing integrated topo III␣
promoter-luciferase DNA, it was noted that the different clones exhibited different degrees of activation by FR. From this observation,
it appears that positional effects are relevant to the FR activation
phenomenon reported here. In contrast to WT cells, stably transfected
AT cell exhibited very low levels of FR activation. These results
imply that the loss of ATM function may have a global effect on
histone acetylation-mediated gene transcription.
On the basis of these observations, decreases in HDAC inhibitormediated induction in AT cells might be explained in two ways. One
is that the ATM protein directly modulates HAT and HDAC activities,
such that overall acetylation is less robust in the absence of a fully
functional ATM gene product. Therefore, reducing HDAC activity
(by addition of FR or TSA) does not culminate in excess acetylation,
because HAT activity is globally less active overall. The other possibility is that the loss of ATM function does not directly influence
histone acetylation but influences the events downstream of histone
acetylation presumably in chromatin. That is, the AT cell will have the
normal levels of acetylation in response to FR or TSA, but somehow
the acetylation fails to result in activation of transcription. Assessment
of FR-induced histone acetylation (Fig. 6) appears to support the
second possibility. There were no substantive differences observed in
histone H3 acetylation between the WT cells and AT cells after the FR
treatment. Although H4 displays a delay in histone acetylation in
response to FR, the final acetylation levels were no different between
the wild type cells and AT cells.
The ATM protein is localized in chromatin and the nuclear matrix
(71). Because ionizing radiation does not change its localization or
amount and because chromatin structure is also abnormal AT cells
(71), it is possible that ATM is required to maintain an appropriate
chromatin structure as a prerequisite for histone acetylation-dependent
gene regulation. Struhl (72) has proposed models for how histone
acetylases and deacetylases selectively affect gene expression. One of
his ideas is that histone acetylations are generally targeted to promoters, and the selection is because of inherent differences in the promoters. For example, acetylation-sensitive promoters are associated
with more tightly packed and/or positioned nucleosomes, and acetylation changes the nucleosome organization to facilitate accessibility
of RNA pol II machinery to promoters. In contrast, acetylationinsensitive promoters are located in less tightly packed and/or positioned nucleosome region, and the organization state of nucleosomes
does not affect the accessibility of pol II machinery. This model may
apply to regulation of p21 or topo III␣. That is, these promoters are
acetylation-sensitive in presence of the normal ATM protein, and loss
of ATM turns these promoters into an acetylation-insensitive mode.
However, because we have not examined the change of local histone
acetylation in response to FR or TSA, for instance, the acetylation
levels of nucleosomes at the p21 promoter, we cannot exclude the
possibilities in which the loss of ATM results in disassembly of
certain HAT complexes on the promoters we tested, which, in turn,
fail to acetylate the histones on those promoters. These acetylated
histones may only account for a small portion of total acetylated
histone; therefore, the change cannot be detected under the conditions
of this study.
A large body of evidence supports the idea that acetylation of
histones activates transcription; however, exactly how the histone
acetylation leads to transcription remains unclear (2). In this study, we
show that the ATM gene product plays a role in this process. Additional understanding of this phenomenon will not only provide an
insight into the mechanism of histone acetylation and transcription but
also open a new angle to understand multiple functions of ATM.
ACKNOWLEDGMENTS
We thank Dr. Michael B. Kastan (St. Jude Children’s Research Hospital,
Memphis, TN) for providing us with ATM-complemented cell lines and Dr.
Bert Vogelstein (The Johns Hopkins University, Baltimore, MD) for the
human p21WAF1 cDNA. We are grateful to Dr. Y. Shiloh for permission to use
the these cell lines, and for helpful discussion. We also sincerely appreciate the
work contributed by and the technical assistance of Dr. Carmen Cantemir.
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Histone Deacetylase Inhibitors Activate p21WAF1
Expression via ATM
Rong Ju and Mark T. Muller
Cancer Res 2003;63:2891-2897.
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