Krüppel-like Factor 4 Mediates p53-dependent
G1/S Cell Cycle Arrest in Response to DNA Damage*
Hong S.
Yoon
,
Xinming
Chen
, and
Vincent W.
Yang
§¶
From the
Division of Digestive Diseases, Department
of Medicine and § Winship Cancer Institute, Emory
University School of Medicine, Atlanta, Georgia 30322
Received for publication, October 29, 2002
 |
ABSTRACT |
The tumor suppressor p53 is required for the
maintenance of genomic integrity following DNA damage. One
mechanism by which p53 functions is to induce a block in the transition
between the G1 and S phase of the cell cycle.
Previous studies indicate that the Krüppel-like factor 4 (KLF4) gene is activated following DNA damage and that such
activation depends on p53. In addition, enforced expression of
KLF4 causes G1/S arrest. The present study examines the requirement of KLF4 in mediating the
p53-dependent cell cycle arrest process in response to DNA
damage. We show that the G1 population of a colon cancer
cell line, HCT116, that is null for the p53 alleles (
/
) was
abolished following
irradiation compared with cells with
wild-type p53 (+/+). Conditional expression of
KLF4 in irradiated HCT116 p53
/
cells
restored the G1 cell population to a level similar to that
seen in irradiated HCT116 p53+/+ cells. Conversely,
treatment of HCT116 p53+/+ cells with small interfering RNA
(siRNA) specific for KLF4 significantly reduced the number
of cells in the G1 phase following
irradiation compared
with the untreated control or those treated with a nonspecific siRNA.
In each case the increase or decrease in KLF4 level because of
conditional induction or siRNA inhibition, respectively, was accompanied by an increase or decrease in the level of
p21WAF1/CIP1. Results of our study indicate that KLF4 is an
essential mediator of p53 in controlling G1/S progression
of the cell cycle following DNA damage.
 |
INTRODUCTION |
The mammalian cell cycle is operationally divided into five
distinct phases: gap 1 (G1), DNA synthesis (S), gap 2 (G2), mitosis (M), and growth arrest phase
(G0), also called quiescence (1). Complex networks of
control mechanisms called "checkpoints" are responsible for the
orderly progression of these events within the cell cycle. Defects in
checkpoint control increase genetic instability, thereby contributing
to uncontrolled proliferation (2). For example, damage to the DNA
elicits a series of signal transduction pathways that result in an
arrest of the cell cycle at various checkpoints (3). Much of the DNA
damage-induced signals are funneled through p53, which directs further
downstream actions that lead to inhibition of G1 to S and
G2 to M transitions, among other events such as apoptosis
(4). Therefore, it is not surprising that p53 is the most
frequently mutated tumor suppressor gene in human cancers (5).
The arrest in the transition between the G1 and S phase of
the cell cycle elicited by p53 requires in part the transcriptional activation of the gene encoding the cyclin-dependent kinase
(Cdk)1 inhibitor
p21WAF1/CIP1 (6, 7). p21WAF1/CIP1 binds to
several G1 cyclin-Cdk complexes and inhibits
phosphorylation of the retinoblastoma susceptibility gene product Rb
(8), a step required for the onset of DNA synthesis (9). Recent
evidence suggests that p21WAF1/CIP1 is also required to
sustain G2 arrest after DNA damage (10). Here,
p21WAF1/CIP1 mediates the function of p53 in response to
DNA damage by inhibiting Cdc2 (11), a Cdk required for entry into
mitosis (12). The proportion of cells that arrests in G1/S
or G2/M depends on the cell type and status of checkpoint
controls in each cell (13).
Although earlier studies indicate that expression of
p21WAF1/CIP1 is the result of direct binding of
p53 to its promoter (14), it is now evident that a myriad of
transcription factors under various physiologic conditions can also
lead to the transcriptional activation of
p21WAF1/CIP1 (15). Among these is the zinc
finger-containing transcription factor, Krüppel-like factor 4 (KLF4), also called gut-enriched Krüppel-like factor or GKLF (16,
17). KLF4 is a member of a rapidly expanding family of mammalian
Krüppel-like factors that exhibit homology to the
Drosophila protein Krüppel (18). Expression of
KLF4 is highly enriched in the postmitotic terminally differentiated epithelial cells of the intestine and epidermis (19,
20). In cultured cells expression of KLF4 is associated with
growth arrest as a result of serum deprivation or contact inhibition
(19, 21). Conversely, enforced expression of KLF4 inhibits
DNA synthesis and results in decreased cell proliferation (19, 22, 23).
These studies suggest that KLF4 is a negative regulator of cell growth.
Recently, it was demonstrated that expression of KLF4 is
also induced by DNA damage and that such induction is dependent on p53
(24). Importantly, KLF4 was shown to physically interact with p53,
resulting in a synergistic activation of the
p21WAF1/CIP1 promoter. Moreover, antisense
inhibition of KLF4 leads to a decreased level of
p21WAF1/CIP1 in response to DNA damage (24), suggesting
that KLF is a potentially important mediator of p53-induced growth
arrest. Indeed, recent studies using an inducible system for
KLF4 indicate that its induction leads to arrest in the
G1/S transition of the cell cycle (25). In the present
study, we further characterize the role of KLF4 in mediating
p53-dependent cell cycle arrest. By manipulating KLF4 expression, we show that KLF4 is essential for the
G1/S cell cycle arrest that results from DNA damage.
 |
EXPERIMENTAL PROCEDURES |
Cell Lines--
The colon cancer cell lines, wild-type and null
for p53, HCT116 p53+/+, and HCT116
p53
/
, respectively, were generous gifts from Dr. Bert
Vogelstein of Johns Hopkins University (10). The cells were cultured in
McCoy's medium supplemented with 10% fetal bovine serum and 1%
penicillin-streptomycin. EcR116 p53
/
cells were
established by stably transfecting pVgRXR (25), which
contains VgEcR and retinoid X receptor that form a receptor for the
insect hormone ecdysone, into the parental HCT116 cell line and
selecting with 100 µg/ml Zeocin (Invitrogen). The level of retinoid X
receptor expression was determined by Western blot analysis.
Irradiation--
irradiation of cultured cells was
performed using a 137Cs
irradiator at 0.8 Gy/min for 15 min, for a total of 12 Gy. Cells were harvested at 24 h after
irradiation for subsequent assays.
Adenovirus Infection--
The recombinant adenovirus containing
green fluorescence protein and KLF4 (AdEGI-KLF4) or green fluorescence
protein alone (AdEGI) were described previously (25, 26). EcR116
p53
/
cells were grown to 40% confluence in 10-cm dishes
and replenished with fresh media containing 2% fetal bovine serum
followed by the addition of 108 plaque-forming units of
recombinant virus per dish. Infected cells were incubated at 37 °C
for 6 h, at which time cells were
-irradiated, and the medium
was changed. Cells were treated with 5 µM ponasterone A
(Invitrogen) for 24 h and then collected for further analysis.
Preparation of siRNA and Transfection--
23-nucleotide
single-stranded RNAs were produced by Integrated DNA Technologies
(Coralville, IA). The small interfering RNA (siRNA) sequences targeting
KLF4 (GenBankTM accession number XM_047517)
correspond to the coding region between nucleotides 121-141 from the
translation initiation site. The complementary single-stranded RNAs
were dissolved in 10 mM Tris-HCl and 1 mM EDTA
(pH 7.0) and annealed in 25 mM KoAc, 10 mM Tris-HCl, and 1 mM EDTA (pH 7.0) by briefly
heating to 70 °C, then incubating for 20 min each at 37 and
23 °C. A nonspecific double-stranded siRNA with identical length was
also generated based on the sequence of an unrelated protein and used
as a control.
HCT116 p53+/+ cells were grown to 40% confluence in 10-cm
dishes,
-irradiated for a total of 12 Gy, and transfected with annealed siRNA using DMRIE-C reagent (Invitrogen) for 6 h as
recommended by the manufacturer. McCoy's medium containing 20%
fetal bovine serum and 2% penicillin-streptomycin was added to each
dish to a final concentration of 10% fetal bovine serum and 1%
penicillin-streptomycin. Cells were harvested 24 h later for
further assays.
Western Blot Analysis--
Cell protein extraction and Western
blot analyses were performed using standard procedures. Protein samples
were mixed with loading buffer (100 mM Tris-HCl, pH 6.8, 2% SDS, 100 mM dithiothreitol, 0.01% bromphenol blue, and
10% glycerol), heated at 100 °C for 5 min, and loaded onto a
SDS-polyacrylamide gel in electrophoresis buffer containing 25 mM Tris-HCl, pH 8.3, 250 mM glycine, and 0.1%
SDS. Protein was then transferred to nitrocellulose membranes using the
Trans-Blot semidry system (Bio-Rad). The membranes were immunoblotted
with primary antibodies against KLF4 (19), p53, p21WAF1/CIP1, or
-catenin (Santa Cruz Biotechnology).
Following incubation with the secondary antibody (horseradish
peroxidase-conjugated goat anti-rabbit IgG, 1:10,000 dilution, Santa
Cruz Biotechnology), KLF4, p53, p21WAF1/CIP1, or
-catenin was visualized with the SuperSignal West Pico
chemiluminescent substrate kit (Pierce).
Cell Cycle Analysis--
Cells were rinsed in Dulbecco's
phosphate-buffered saline (Mediatech), trypsinized, resuspended in
McCoy's medium containing 10% fetal bovine serum and 1%
penicillin-streptomycin, collected by centrifugation, washed with
Dulbecco's phosphate-buffered saline, again collected by
centrifugation, resuspended in 70% ethanol, and fixed at
20 °C
overnight. Cells were pelleted again by centrifugation and re-suspended
in a staining solution containing 50 µg/ml propidium iodide, 50 µg/ml RNase A, 0.1% Triton X-100, and 0.1 mM EDTA for 30 min. Flow cytometry was performed on a FACSCalibur (BD Biosciences) cytometer.
 |
RESULTS |
G1/S Arrest Depends on p53 in HCT116 Cells
following
Irradiation--
Both HCT116 p53+/+ and
p53
/
cells exhibited comparable cell cycle profiles
before irradiation (Fig. 1, A,
B, and E). Following 12 Gy of
irradiation,
HCT116 p53+/+ cells demonstrated a normal cell cycle arrest
pattern, with ~15% of cells in G1, ~80% cells in
G2, and a significantly reduced S population (Fig. 1,
C and F). However, HCT116 p53
/
cells exhibited an abnormal cell cycle pattern after
irradiation,
with ~90% of the cells in G2 and few remaining in either
G1 or S (Fig. 1, D and F). Consistent with the effect of
irradiation on the cell cycle of HCT116
p53+/+ cells, protein levels of KLF4 and
p21WAF1/CIP1 were both significantly increased in response
to an increase in p53 protein levels (Fig.
2, lanes 1 and 2).
In contrast, no induction in the level of either KLF4 or
p21WAF1/CIP1 was observed following
irradiation in
HCT116 p53
/
cells (Fig. 2, lanes 3 and
4). These results suggest that at least part of the cell
cycle arrest caused by
irradiation is a result of
p53-dependent activation of KLF4 and
p21WAF1/CIP1.

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Fig. 1.
The effect of irradiation on HCT116 p53+/+ and HCT116
p53 / cells. In panels A through
D flow cytometric analyses of HCT116 p53+/+ and
HCT116 p53 / were performed 24 h after 0 or 12 Gy of
irradiation. Cells were stained with propidium iodide, and DNA
content was analyzed by flow cytometry. The DNA content of haploid and
diploid cells is designated 2n and 4n,
respectively. Panels E and F show the means and
standard deviations of percentage of the G1, S, and
G2/M populations from five independent experiments in
non-irradiated and irradiated cells, respectively. Open bars
represent HCT116 p53+/+, and closed bars
represent HCT116 p53 / . 15,000 cells were analyzed in
each experiment. *, p < 0.05 compared with HCT116
p53+/+ cells.
|
|

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Fig. 2.
Western blot analysis of p53, KLF4, and
p21WAF1/CIP1 in response to irradiation. The levels of p53, KLF, and
p21WAF1/CIP1 were determined by Western blot analysis in
HCT116 p53+/+ (lanes 1 and 2) and
HCT116 p53 / (lanes 3 and 4)
without irradiation (lanes 1 and 3) and 24 h
after 12 Gy of irradiation (lanes 2 and
4).
|
|
Inducible Expression of KLF4 in HCT116 p53
/
Cells
Restores G1 Peak--
The failure of
irradiation to
induce expression of KLF4 and
p21WAF1/CIP1 in HCT116 p53
/
cells
correlated with the reduction in G1 and S populations
(Figs. 1 and 2). This suggests that activation of KLF4, with
consequent activation of p21WAF1/CIP1, may be
necessary for the accumulation of cells in G1. To test this
hypothesis, we established a stable HCT116 p53
/
cell
line that expressed the receptors for the insect nuclear hormone,
ecdysone, and its partner, retinoid X receptor (25). This cell line,
called EcR116 p53
/
, was infected with the recombinant
adenovirus AdEGI or AdEGI-KLF4 (25) that contained enhanced green
fluorescence protein as a control or enhanced green fluorescence
protein plus KLF4, respectively. Following infection, cells were
-irradiated or not and then treated with the inducer, ponasterone A,
or vehicle alone for 24 h before being harvested for cell cycle
analysis. As seen in Fig. 3, treatment of
AdEGI-KLF4-infected cells with ponasterone A without irradiation
resulted in a statistically significant increase in the G1
population and a decrease in the G2/M population (Fig. 3,
C, D, and J), whereas AdEGI-infected cells without irradiation and treated with ponasterone A had no effect
on the cell cycle when compared with untreated cells (Fig. 3,
A, B, and I). Cells infected with
AdEGI followed by irradiation showed G2/M arrest in
the absence or presence of ponasterone A (Fig. 3, E,
F, and K) as did cells infected with AdEGI-KLF4
and irradiated without any ponasterone A treatment (Fig. 3,
G and L). In contrast, upon the addition of
ponasterone A, AdEGI-KLF4-infected and irradiated cells had a
statistically significant increase in the G1 population
(Fig. 3, H and L). This finding is reminiscent of
the G1/S arrest seen in HCT116 p53+/+ cells following
irradiation (compare Figs. 3H and 1C).

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Fig. 3.
The effect of inducible KLF4
expression on cell cycle of p53 / cells
following irradiation. EcR116
p53 / cells were infected with AdEGI or AdEGI-KLF4 and
then irradiated with 12 Gy of radiation (panels E,
F, G, and H) or not (panels
A, B, C, and D). Cells were then
treated with vehicle alone (panels A, E,
C, and G) or ponasterone A (PA)
(panels B, F, D, and H) for
24 h before being harvested for flow cytometric analysis.
Panels I-L show the means and standard deviations of
percent cells in the three phases of the cell cycle in 5 independent
experiments. 15,000 cells were analyzed per experiment. *,
p < 0.05 compared with uninduced cells.
|
|
Fig. 4 shows that only cells infected by
AdEGI-KLF4 and induced with ponasterone A (lanes 11 and
12) had appreciable amounts of KLF4. The increase in the
KLF4 level correlated with an increase in the p21WAF1/CIP1
level, a finding consistent with our previous observation that KLF4 is
an activator of p21WAF1/CIP1 expression (25). The
combined results of Figs. 3 and 4 indicate that the inducible
expression of KLF4 in irradiated cells lacking p53 restores the characteristic G1/S arrest in
cells with wild type p53 following irradiation. This finding
indicates that KLF4 is necessary and sufficient in mediating the
G1 cell cycle effect of p53 following DNA damage.

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Fig. 4.
Western blot analysis of KLF4 and
p21WAF1/CIP1 in EcR116 p53 /
cells. The levels of KLF4 and p21WAF1/CIP1 were
determined by Western blot analysis in EcR116 p53 / cells
not infected with any virus (lanes 1-4), infected with
AdEGI (lanes 5-8) or with AdEGI-KLF4 (lanes
9-12), followed by 0 (lanes 1, 3,
5, 7, 9, and 11) or 12 Gy
(lanes 2, 4, 6, 8,
10, and 12) of irradiation and induced with
ponasterone A (PA) (lanes 3, 4,
7, 8, 11, and 12) or
vehicle alone (lanes 1, 2, 5,
6, 9, and 10).
|
|
Small Interfering RNA Targeting KLF4 mRNA Abolishes
G1 Arrest in
-irradiated HCT116 p53+/+
Cells--
Recently, Tuschl and co-workers (27, 28) demonstrated
that RNA interference can be provoked in mammalian cell lines through the introduction of siRNA. The mediators of sequence-specific mRNA
degradation are 21-23-nucleotide siRNA duplexes that trigger specific
gene silencing in mammalian somatic cells without activation of the
unspecific interferon response (27-29). To determine whether we could
"knock down" KLF4 expression using siRNA, we synthesized a 23-nucleotide siRNA duplex specific for KLF4 to transfect
HCT116 p53+/+ cells with or without irradiation. As seen in
Fig. 5, siRNA for KLF4
significantly reduced the level of KLF4 in response to
irradiation
when compared with untransfected or mock-transfected cells (lanes
2, 4, 6, and 8). In contrast, a
control nonspecific siRNA failed to abrogate the DNA damage-induced
synthesis of KLF4 (lanes 10 and 12). Again, there
was a corresponding reduction in the level of p21WAF1/CIP1
in response to
irradiation in KLF4 siRNA-treated cells
(lanes 6 and 8). Importantly, KLF4
siRNA but not nonspecific siRNA abolished the G1 population
in cells upon
irradiation (Fig. 6,
lanes F, G, H, I, and
J). These results complement those from the preceding sections and provide strong evidence that KLF4 is an essential factor
in mediating p53-dependent G1 arrest in
response to DNA damage.

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Fig. 5.
The effect of KLF4 siRNA on protein levels of
p53, KLF4, and p21WAF1/CIP1 in HCT116 p53+/+ cells
following irradiation. The levels of
p53, KLF4, and p21WAF1/CIP1 were determined in
-irradiated (even lanes) or non-irradiated (odd
lanes) HCT116 p53+/+ cells that were untransfected
(lanes 1 and 2), mock-transfected (lanes
3 and 4), transfected with 2 (lanes 5 and
6) or 4 µg (lanes 7 and 8) of
KLF4-specific siRNA, or transfected with 2 (lanes
9 and 10) or 4 µg (lanes 11 and
12) of nonspecific siRNA. Cells were harvested 24 h
later for Western blot analysis of protein levels.
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Fig. 6.
The effect of KLF4 siRNA on the cell cycle in
HCT116 p53+/+ cells following irradiation. Flow cytometric analyses were performed in
HCT116 p53+/+ cells that were non-irradiated (panels
A-E) or irradiated (panels F-J) followed by mock
transfection (panels A and F), transfection with
2 (panels B and G) or 4 µg (panels C
and H) of KLF4-specific siRNA, or transfection
with 2 (panels D and I) or 4 µg (panels
E and J) of nonspecific siRNA. Cells were studied for
24 h following irradiation. Panels K and L
show the means and standard deviations of percent cells in
G1 phase for each of the treatments in five independent
experiments. 15,000 cells were analyzed in each experiment. *,
p < 0.05 compared with mock-transfected cells.
|
|
 |
DISCUSSION |
Cell cycle progression is regulated by checkpoint controls, which
function to safeguard the integrity of the genome. Activation of DNA
integrity checkpoints occurs through the detection of damaged or
unreplicated DNA and is in effect until DNA damage has been repaired
(30). The checkpoint that arises after DNA damage can activate during
G1, S, or G2 (3, 31). Arrest in G1
permits repair prior to replication, whereas arrest in S or
G2 permits repair of the genome before mitotic segregation.
The p53 tumor suppressor has been shown to be integral to both the
G1 (32, 33) and G2 (34, 35) DNA damage
machinery. This was supported by the results in Fig. 1, which showed
that HCT116 p53+/+ cells arrested at either G1
or G2/M after
irradiation as expected for cells with
intact checkpoint function. The resultant activation of p53 because of
irradiation was accompanied by a significant increase in the level
of KLF4 and p21WAF1/CIP1 (Fig. 2) in a manner similar to
the previously observed response of fibroblasts subjected to DNA damage
caused by methyl methanesulfonate (24). HCT116 p53
/
cells, in contrast, showed no induction of either KLF4 or
p21WAF1/CIP1 by
irradiation and arrested only in
G2/M (Fig. 1). The latter result was consistent with that
from a previous study, which also demonstrated that p53 was necessary
to sustain G2 arrest (10).
Recent studies indicate that the G1 checkpoint control
after DNA damage consisted of two steps (31). The first step is a rapid
and p53-independent induction of the G1 checkpoint. It is a
result of rapid redistribution of p21WAF1/CIP1 from cyclin
D1-Cdk4/6 complexes to cyclin E-Cdk2 complexes, which are inhibited by
p21WAF1/CIP1 (36, 37). The second step involves the
post-translational modifications of p53 by upstream protein kinases,
including ataxia telangiectasia mutated/ataxia telangiectasia and Rad3
related and Chk1/Chk2 (38, 39), which results in p53 activation
and subsequent transcriptional induction of
p21WAF1/CIP1 (31). Several lines of evidence
suggest that KLF4 is involved in the p53-dependent
induction of p21WAF1/CIP1. First, p53 mediates the
transcriptional induction of KLF4 in response to DNA damage
(24). Second, the induction in KLF4 precedes that in
p21WAF1/CIP1 following DNA damage (24). Third,
KLF4 binds to a specific cis-element in the proximal promoter of the
p21WAF1/CIP1 gene and activates the promoter (24).
Fourth, p53 and KLF4 physically interact and cause a synergistic
induction in p21WAF1/CIP1 gene expression (24).
The importance of KLF4 in mediating the transcriptional induction of
p21WAF1/CIP1 is further demonstrated by the
observation that p53 fails to activate the
p21WAF1/CIP1 promoter if the KLF4 response element
in the promoter is mutated (24).
In addition to the biochemical evidence supporting a crucial role for
KLF4 in mediating the transcriptional induction of
p21WAF1/CIP1 by p53, the present study provides
the genetic evidence to further substantiate the significance of KLF4
in p53-mediated G1 arrest caused by DNA damage.
Specifically, the conditional induction of KLF4 in
-irradiated HCT116 p53
/
cells restored the
G1 population of cells that are normally present in
irradiated HCT116 p53+/+ cells (Fig. 3). Conversely,
inhibition of KLF4 expression in irradiated HCT116
p53+/+ cells resulted in an abolishment of the
G1 peak in a manner that resembles the consequence of
irradiation of HCT116 p53
/
cells (Fig. 6). In each case,
the induction or inhibition of KLF4 expression was
accompanied by a corresponding increase or decrease, respectively, in
the level of p21WAF1/CIP1 (Figs. 4 and 5). Coupled with the
findings from our previous study, which demonstrated that inducible
expression of KLF4 causes a G1/S cell cycle
arrest (25), it is highly likely that KLF4 serves a pivotal role in
mediating the G1 checkpoint function of p53 in response to
DNA damage.
In addition to its effect on the G1/S checkpoint, p53 also
regulates the G2/M transition in response to DNA damage
(11). Part of the mechanism by which p53 inhibits the G2
checkpoint involves inhibition of Cdc2, the
cyclin-dependent kinase required to enter mitosis (12).
Binding of Cdc2 to cyclin B1 is required for its activity, and
repression of the cyclin B1 gene by p53 contributes to the
blocking of entry into mitosis (40, 41). p53 also represses expression
of the Cdc2 gene (42, 43) to help ensure that cells do not
escape from the initial block. Moreover, several of the transcriptional
targets of p53 can inhibit Cdc2, including p21WAF1/CIP1,
14-3-3
, and Gadd45 (44-46). Therefore, it is of great interest to
note that a recent analysis of KLF4 target genes by cDNA
microarrays showed that KLF4 activates expression of
14-3-3
, in addition to
p21WAF1/CIP1, and represses expression of
Cdc2 (47). Whether KLF4 is
also involved in mediating the G2 checkpoint function of
p53 in response to DNA damage is currently being determined.
 |
ACKNOWLEDGEMENT |
We thank Dr. Bert Vogelstein for kindly
providing the HCT116 p53+/+ and
/
cell lines.
 |
FOOTNOTES |
*
This work was supported in part by Grants DK52230 and
CA84197 from the National Institutes of Health.The costs of publication of this
article were defrayed in part by the
payment of page charges. The article
must therefore be hereby marked
"advertisement" in accordance with 18 U.S.C. Section
1734 solely to indicate this fact.
¶
Recipient of a Georgia Cancer Coalition Distinguished Cancer
Clinician Scientist award. To whom correspondence should be addressed: 201 Whitehead Biomedical Research Bldg., Emory University School of
Medicine, 615 Michael St., Atlanta, GA 30322. Tel.: 404-727-5638; Fax:
404-727-5767; E-mail: vyang@emory.edu.
Published, JBC Papers in Press, November 8, 2002, DOI 10.1074/jbc.M211027200
 |
ABBREVIATIONS |
The abbreviations used are:
Cdk, cyclin-dependent kinase;
EcR, ecdysone receptor;
KLF4, Krüppel-like factor 4;
siRNA, small interfering RNA;
Gy, gray;
AdEGI, recombinant adenovirus containing green fluorescence
protein.
 |
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