From the Department of Radiation Medicine and
¶ Markey Cancer Center, University of Kentucky,
Lexington, Kentucky 40536 and the
Department of Pathology,
Washington University, St. Louis, Missouri 63130
Received for publication, September 15, 2000, and in revised form, October 13, 2000
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ABSTRACT |
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In this study, we sought to investigate the
mechanism of the proapoptotic function of Egr-1 in
relation to p53 status in normal isogenic cell backgrounds by using
primary MEF cells established from homozygous
(Egr-1 The apoptotic pathways consist of an early component that includes
molecular events specific for an inducer or a group of inducers and of
downstream effector components common to diverse apoptotic signals (1).
Apoptosis has also been reported in a variety of experimental tumor
systems following exposure to radiation (2, 3). Ionizing radiation
alters the expression of specific genes, the products of which may
contribute to the events leading to apoptotic cell death. Ionizing
radiation exposure is associated with activation of certain
immediate-early genes that function as transcription factors (4). These
include members of jun or fos and early growth
response (EGR)1 gene families
(5, 6).
The Egr gene family includes Egr-1 (7),
Egr-2 (8), Egr-3 (9), Egr-4 (10), and
the tumor suppressor, Wilms' tumor gene product, WT1 (11, 12). The
Egr family shows a high degree of homology in the amino
acids constituting the zinc finger domain and binds to the same GC-rich
consensus DNA sequence (13, 14). The Egr-1 gene product,
EGR-1, is a nuclear protein that contains three zinc fingers of the
C2H2 subtype (15, 16). Structure-function mapping studies on EGR-1 protein suggest that the amino acids constituting the zinc finger motif confer DNA binding function, whereas
the NH2- terminal amino acids confer transactivation
function (16, 17). More recent studies have found that sequences
diverging from the consensus may also bind EGR-1 (18, 19), thus having a broader spectrum of potential target genes. It is interesting to note
that within this family of transcription factors, EGR-1 was found to be
a positive activator of transcription, whereas WT1 is a transcriptional
repressor, both acting via binding to the same GC-rich consensus
sequence in reporter constructs (20-22). Depending on the cell type,
EGR-1 may behave as a positive or negative regulator of gene
transcription (16, 23, 24). The EGR-1 GC-rich consensus target
sequence, 5'-GCG(T/G)GGGCG-3' or 5'-TCC(T/A)CCTCCTCC-3' (25), has been
identified in the promoter regions of the following: (a)
transcription factors, such as MYC and NUR77; (b) growth
factors or their receptors, such as transforming growth factor- It has also been speculated that x-ray induction of PDGF, transforming
growth factor- On the other hand, wild type p53 has been shown to be
functionally necessary for growth inhibition and apoptosis following exposure to ionizing radiation, and p53 mutations have been
reported to increase resistance to apoptosis (34). In our previous
report, using melanoma cells, which contain wild type p53, a
dose-dependent increase in EGR-1 expression with
dose-dependent growth inhibition was observed when exposed
to ionizing radiation (35). Transfectant melanoma cells stably
expressing the dominant-negative mutant protein of EGR-1 showed
significantly reduced (<50%) sensitivity to radiation-inducible
growth inhibition, and this resistance was found to be
dose-dependent. These observations suggest that the EGR-1
induction is involved in the regulation of radiation-inducible apoptosis despite the presence of wild type p53. Recently, we used a
p53 null prostate cancer cell line (PC-3), which was found to be
moderately resistant to ionizing radiation-inducible apoptosis (36).
Western blot analysis and immunocytochemistry studies indicate that
EGR-1 is induced in the PC-3 cells by ionizing radiation. Experiments
with the Egr-1 dominant-negative mutant or Egr-1
overexpression suggest that Egr-1 function is required for
the radiation-inducible apoptosis. Despite the absence of wild type
functional p53 protein, the transfected cells expressing the
dominant-negative mutant of EGR-1 were resistant to ionizing radiation,
and cells overexpressing EGR-1 protein were sensitive to ionizing
radiation. Our findings strongly suggested that the radiation-induced
apoptotic response in PC-3 cells is elicited through up-regulation of
TNF- Cell Culture--
Primary cultures of mouse embryonic fibroblast
(MEF) cells from normal mice at passage 3 (kindly provided by Dr. Tyler
Jacks, Howard Hughes Medical Institute) labeled as p53+/+
were assumed to have two normal alleles of Egr-1 wild type
(Egr-1+/+) gene. Primary MEF cultures of cells
at passage 0 from homozygous (Egr-1 Plasmid Constructs--
The plasmid CMV-EGR-1, which encodes
full-length EGR-1 protein, contains EGR-1 cDNA downstream of the
CMV promoter in the vector pCB6+ (21). Plasmid
pCMV-WT1-EGR-1, which encodes a dominant-negative mutant of EGR-1,
contains a WT1-EGR-1 chimera downstream of the CMV promoter in the
vector pCB6+ (21). The reporter construct, EBS-CAT,
contains three EGR-1-binding sites (CGCCCCCGC) placed in tandem
upstream of a minimal c-fos promoter and CAT cDNA. The
p53-CAT construct (pAA-CAT), which contains 337-base pair
(AvaII-AvaII) fragment ( DNA Transfection and CAT Assays--
Transient transfections
were performed by the calcium phosphate coprecipitation method as
described previously (36). CAT assays were performed by thin layer
chromatography as described previously (36).
Irradiation--
A 100-kV industrial x-ray machine (Phillips,
Netherlands) was used to irradiate the cultures at room temperature.
The dose rate with a 2-mm aluminum plus 1-mm beryllium filter was 3.85 Gy at a focus-surface distance of 20 cm.
Quantitation of Apoptosis--
Apoptosis was quantified by TUNEL
staining and flow cytometry. The ApopTag in situ apoptosis
detection kit (Oncor, Gaithersburg, MD), that detects DNA strand breaks
by terminal transferase-mediated dUTP-digoxigenin nick end labeling
(TUNEL) was used as described (36). Briefly, cells were seeded in
chamber slides, and the next day they were exposed to a 5-Gy dose of
radiation. After 24 h, the DNA was tailed with digoxigenin-dUTP
and conjugated with an anti-digoxigenin fluorescein. The specimen was
counter stained with propidium iodide and antifade. The stained
specimen was observed in triple band-pass filter using Nikon-microphot epifluorescence microscope. To determine the percentage of cells showing apoptosis, four experiments in total were performed, and ~1000 cells were counted in each experiment. For flow cytometry, cells were lifted by using nonenzymatic cell dissociation medium (Sigma), washed with phosphate-buffered saline, stained with Hoechst (Ho342) and merocyanine (MC540), and analyzed by flow cytometry using a
FACStar Plus cell sorter as described (36).
32P-Reverse Transcriptase-Polymerase Chain Reaction
(32P-RT-PCR) of p53 and Its Target Genes--
Total RNA
was isolated from untreated and irradiated
Egr-1+/ Western Blot Analysis--
Total protein extracts from untreated
and irradiated cells at various time intervals were subjected to
Western blot analysis as described (35), using anti-EGR-1 antibody
(sc-110) (Santa Cruz Biotechnology), anti-p53 antibody (pAb240)
(sc-99), anti-MDM2 antibody (PharMingen 65101A), anti-Rb antibody
(PharMingen 14001 A), or for loading control the anti- Immunoprecipitation--
Cells were lysed with triple detergent
lysis buffer (50 mM Tris-Cl, pH 8.0, 150 mM
NaCl, 0.02% sodium azide, 0.1% SDS, 100 µg/ml phenylmethylsulfonyl
fluoride, 1 µg/ml aprotinin, 1% Nonidet P-40, 0.5% sodium
deoxycholate), and 1 mg of lysates was incubated with antibodies
(either anti-MDM2 antibody, PharMingen 65101A, or goat polyclonal
anti-p53 antibody FL-393-G) as indicated. The antibody complexes were
isolated using protein A/G-agarose beads (Santa Cruz Biotechnology) and
washed three times with phosphate-buffered saline. The
immunoprecipitated protein with beads were boiled in SDS sample buffer,
and the supernatants were analyzed on SDS-polyacrylamide gel and
subjected to Western blot analysis using anti-Rb antibody or anti-MDM2 antibody.
Ionizing Radiation Induces EGR-1 Protein That Transactivates via
the GC-rich Binding Site in Egr-1+/
To ascertain the EGR-1-dependent transactivation process in
Egr-1 Ionizing Radiation Caused Enhanced Cell Death in
Egr-1+/ High Basal Levels and Lack of Induction of p53,
p21waf1/cip1, mdm-2, and bax mRNA by Radiation in
Egr-1 Ionizing Radiation Caused Down-regulation of p53 Protein in
Egr-1 Transfection of CMV-EGR-1 in Egr-1
We further performed p53-CAT reporter assays to understand whether the
down-regulation of p53 protein after radiation was due to loss of
EGR-1-mediated transactivation in
Egr-1 Overexpression of CMV-EGR-1 in p53
In p53+/+ MEF cells, overexpression of EGR-1 caused
significant induction of cell death after 48 h when compared with
p53+/+ cells overexpressing vector alone. However,
p53+/+ cells overexpressing dominant-negative mutant EGR-1
showed reduction in cell death when compared with p53+/+
cells overexpressing vector alone (Fig. 6A).
Interestingly, overexpression of EGR-1 protein in
p53 Lack of a Hypophosphorylated Form of Rb Protein Led to
MDM2-mediated p53 Degradation in Egr-1
First, to ascertain EGR-1-mediated induction of Rb, we performed
Western blot analysis for Rb protein expression levels and Rb-CAT
reporter assays in untreated and irradiated
Egr-1+/
Low levels of hypophosphorylated forms of Rb and low levels of MDM2
after radiation were evident in
Egr-1 Exposure to ionizing radiation is associated with the formation of
reactive oxygen intermediates causing direct damage to DNA (44). These
reactive oxygen intermediates target the sequence CC(A/T)6GG to mediate the activation of EGR-1 (4). Previous studies from our laboratory (35) have suggested that despite the
presence of wild type p53 background, inhibition of the expression or
function of EGR-1 causes a diminution of radiation-induced growth
inhibition in melanoma cells. In the absence of p53, radiation-induced apoptosis of prostate cancer cells was found to be mediated by EGR-1
via TNF- In this study, in contrast to Egr-1+/ The tumor suppressor gene p53 is a central mediator of
apoptotic pathways in diverse model systems (45-48). The p53 protein can cause transcriptional up-regulation of a number of downstream genes, such as mdm-2, p21waf1/cip1, bax,
fas/apo1, insulin-like growth factor-binding protein-3, which are implicated in growth inhibition and apoptotic cell death (46-48). In this study, it was found that mRNA levels of
p53, p21waf1/cip1, mdm-2, and
bax were elevated after irradiation in
Egr-1+/ Radiation caused degradation of p53 protein in
Egr-1 A marginal induction of radiation-induced apoptosis observed in
p53 p53 can bind to the promoter region of MDM2 and activate its
transcription, forming an autoregulation loop between the expression and function of p53 and MDM2 (49). It is also reported that MDM2-p53
interaction can target p53 for degradation (43). Rb can regulate the
apoptotic function of p53 through binding to MDM2, thus preventing
MDM2-mediated degradation of p53 (42). Rb can also prevent MDM2 from
inhibiting p53-mediated apoptosis. In addition, Rb can protect p53 from
MDM2-mediated degradation by forming a trimeric complex with p53 via
binding to MDM2 (42). To understand further the mechanism of p53
degradation in irradiated Egr-1/
) and heterozygous
(Egr-1+/
) Egr-1 knock-out mice.
Ionizing radiation caused significantly enhanced apoptosis in
Egr-1+/
cells (22.8%; p < 0.0001) when compared with
Egr-1
/
cells (3.5%). Radiation
elevated p53 protein in Egr-1+/
cells in 3-6
h. However, in Egr-1
/
cells,
the p53 protein was down-regulated 1 h after radiation and was
completely degraded at the later time points. Radiation elevated the
p53-CAT activity in Egr-1+/
cells but not in
Egr-1
/
cells. Interestingly,
transient overexpression of EGR-1 in p53
/
MEF cells caused marginal induction of radiation-induced apoptosis when
compared with p53+/+ MEF cells. Together, these results
indicate that Egr-1 may transregulate p53, and both EGR-1
and p53 functions are essential to mediate radiation-induced apoptosis.
Rb, an Egr-1 target gene, forms a trimeric complex with p53
and MDM2 to prevent MDM2-mediated p53 degradation. Low levels of Rb
including hypophosphorylated forms were observed in
Egr-1
/
MEF cells before and
after radiation when compared with the levels observed in
Egr-1+/
cells. Elevated amounts of the
p53-MDM2 complex and low amounts of Rb-MDM-2 complex were observed in
Egr-1
/
cells after radiation.
Because of a reduction in Rb binding to MDM2 and an increase in MDM2
binding with p53, p53 is directly degraded by MDM2, and this leads to
inactivation of the p53-mediated apoptotic pathway in
Egr-1
/
MEF cells. Thus, the
proapoptotic function of Egr-1 may involve the mediation of
Rb protein that is essential to overcome the antiapoptotic function of
MDM2 on p53.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
1,
TNF-
, PDGF-A (26), PDGF-B (27), insulin-like growth factor-II,
fibroblast growth factor-
, or epidermal growth factor receptor (6,
7, 28, 29); (c) cell cycle regulators such as the
retinoblastoma susceptibility gene Rb (30), cyclin D1 (31),
c-Ki-ras (26), and p53 (32); and (d)
thymidine kinase, an enzyme crucial in DNA biosynthesis (18) and MDR-1 (33).
1, and TNF-
may be regulated by Egr-1 and c-jun (6). Apart from being a potential transcriptional regulator, Egr-1 has a radiation-inducible promoter. Through
these distinct induction pathways, Egr-1 has been linked to
signaling events initiating cell phenotypic response to radiation injury.
protein via EGR-1-mediated transactivation. Thus, EGR-1 is an
important mediator of radiation responsiveness irrespective of p53
functional status. However, in a recent report, it was found that EGR-1
protein transactivates the promoter of p53 gene and
up-regulates p53 mRNA and protein levels in response to apoptotic
stimuli (32). This prompted us to investigate further the interactive
role of Egr-1 with p53 during the process of apoptosis. We
sought to investigate this mechanism in a normal cell background using
isogenic normal primary culture cells derived from mouse embryonic
fibroblasts (MEF) with varied genomic status for Egr-1 gene
(cells with both intact Egr-1 alleles,
Egr-1+/+; cells with homozygous deletion of
Egr-1 alleles,
Egr-1
/
; and heterozygous
deletion of one Egr-1 allele,
Egr-1+/
). Based on findings from these
isogenic normal cells with varied genomic status of Egr-1,
we suggest that EGR-1 function is necessary for enhanced sensitivity to
radiation-induced apoptosis and that the radiation-induced proapoptotic
function of Egr-1 is directly mediated by the target genes
p53 and Rb.
MATERIALS AND METHODS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
/
) and heterozygous
(Egr-1+/
) Egr-1 knock-out mice (37)
were grown in Dulbecco's modified Eagle's medium supplemented with
1% glutamine, 10% fetal bovine serum, and 1% penicillin/streptomycin
at 37 °C and 5% CO2. Primary MEF cells at passage 3 containing homozygous deletion of wild type p53 gene
(p53
/
) established from p53 knock-out mice
were also grown in Dulbecco's modified Eagle's medium (kindly
provided by Dr. Tyler Jacks, Howard Hughes Medical Institute).
441 to
104) of p53 promoter placed in front of CAT cDNA (38), was
kindly provided by Dr. Moshe Oren, Wiezmann Institute of Science,
Israel. The EGR-1-binding site (TCC)3T(TCC) on pAA-CAT was
located at
44 to
32. The Rb promoter region from
1343 to
1135
was generated from mouse genomic DNA template by PCR. The sense primer
(5'-TTTTTCTAGACGAGCCTCGCGGACGTGA-3') and antisense primer
(5'-AAAAAAGCTTCATGACGCGCACGCGGGC-3') contained built-in-sites (underlined) for XbaI and HindIII,
respectively, and they generated a 236-base pair fragment of Rb
promoter. The 236-base pair fragment of Rb promoter was
cloned in pG-CAT, a vector for CAT reporter (Rb-CAT). The control pCAT
reporter vector was purchased from Promega.
and
Egr-1
/
cells at various time
intervals using TRIzol reagent (Life Technologies, Inc.). One µg of
total RNA was reverse-transcribed into cDNA using oligo(dT) primers
and reverse transcriptase in a 40-µl reaction mix as described
previously (35). Radiation-induced mRNA expression of
p53, p21waf1/cip1, mdm-2, and
bax were analyzed by PCR. PCR was performed by using the
products of reverse transcription reaction and the upstream and
downstream primers flanking the p53, p21waf1/cip1,
mdm-2, and bax genes (Table
I) and
-actin gene as an internal control.
Sequences of primers used for 32P RT-PCR analysis
-actin
antibody (Sigma). The bound immune complexes were detected using the
chemiluminescence method (Santa Cruz Biotechnology).
RESULTS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
and
Egr-1+/+ MEF Cells--
To determine whether radiation
causes induction of EGR-1 protein in Egr-1+/
and Egr-1+/+ MEF cells, whole cell protein
extracts were prepared from the cells at different time intervals after
exposure to a 5-Gy dose of ionizing radiation and subjected to Western
blot analysis. As shown in Fig.
1A, no detectable basal level
of EGR-1 protein was found in untreated
Egr-1+/
and
Egr-1
/
cells. After exposure to
5-Gy dose of radiation, EGR-1 protein was induced at 30 min (10-fold)
after the exposure (Fig. 1A) in Egr-1+/
cells. However, this induction was
absent in Egr-1
/
cells.
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Fig. 1.
EGR-1 is induced by ionizing radiation.
A, EGR-1 protein induction detected by Western blot
analysis. Whole cell protein extracts were prepared from
Egr-1+/ and
Egr-1
/
cells that were left
untreated (UT) or exposed to a 5-Gy dose of ionizing
radiation for various time intervals (in hours) and then subjected to
Western blot analysis for EGR-1 or
-actin. The blot was subsequently
probed with an antibody for EGR-1 or
-actin. B, ionizing
radiation and CMV-EGR-1 transactivate EBS-CAT reporter construct
containing three tandem repeats of EGR-1-binding sites. MEF cells were
transiently cotransfected with 4 µg of EBS-CAT or 4 µg of
CMV-EGR-1. Next, the cells were either left unexposed or exposed to a
5-Gy dose of ionizing radiation, and CAT activity was assayed and
expressed as percent conversion of [14C]chloramphenicol
to acetylated forms.
/
,
Egr-1+/
, and Egr-1+/+
MEF cells, we performed transient transfections with the following: (a) only reporter construct EBS-CAT that contains three
tandem EGR-1-binding sites; (b) EBS-CAT and an EGR-1
expression construct CMV-EGR-1; or (c) EBS-CAT and then
exposed the cells to ionizing radiation. As seen in Fig. 1B,
CAT activity was completely absent in basal and irradiated
Egr-1
/
cells, whereas transient
transfection with CMV-EGR-1 elevated the CAT activity. In
Egr-1+/
and Egr-1+/+
cells, ionizing radiation increased the relative CAT activity in an
allelic dose-dependent manner. Similarly,
Egr-1+/+ cells showed slightly higher basal CAT
activity as compared with Egr-1+/
cells.
However, the CMV-EGR-1 construct caused an increase in CAT reporter
activity irrespective of endogenous Egr-1 allelic status
(Fig. 1B). These results confirmed that the EGR-1 protein is
necessary for the transactivation of target genes containing the
EGR-1-binding sequence.
Cells--
MEFs
(Egr-1
/
and
Egr-1+/
cells) were left untreated or
irradiated at 5-Gy dose of ionizing radiation. TUNEL staining and flow cytometry were performed to determine the incidence of apoptosis. By
TUNEL assay, the incidence of apoptosis after 24 h of radiation was 3.5% in Egr-1
/
cells and
22.8% in Egr-1+/
cells (Fig.
2A). By flow cytometry assay
using MC540 and Hoechst 342 staining, the incidence of apoptosis after
48 h of radiation was 6.2% in
Egr-1
/
cells and 53% in
Egr-1+/
cells (Fig. 2B). Thus,
ionizing radiation caused significantly enhanced apoptosis in
Egr-1+/
cells (p < 0.0001)
when compared with Egr-1
/
cells
as demonstrated by TUNEL and flow cytometry assays. These observations
suggest that despite the presence of wild type functional p53 gene in this normal cell background, MEFs with
homozygous deletion of Egr-1 were resistant to ionizing
radiation-inducible apoptosis.
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Fig. 2.
Radiation-induced apoptosis in
Egr-1+/ and
Egr-1
/
MEF cells. A,
quantification of apoptosis by TUNEL assay. Apoptosis was quantified by
TUNEL. To determine the percentage of cells showing apoptosis, a total
of 1000 cells were counted for each experiment. Background levels in
untreated cells were normalized over those in treated cells. Data
represents a mean of two experiments. The error bars
represent S.D. B, quantitation of apoptosis by Hoechst 33342 (Ho342) and merocyanine 540 (MC540) staining.
Cells were irradiated at 5 Gy and after 48 h stained with Hoechst
33342 and merocyanine 540. The gates were set so as to analyze cell
cycle and apoptosis stages as described previously (37). Hoechst 33342 is a DNA-specific dye that measures DNA content, and merocyanine 540 binds to membrane phospholipids that are exposed on the outside of the
membrane during the process of apoptosis. These two dyes separate five
distinct populations of tumor cells as follows: viable resting cells,
2n DNA content and merocyanine 540-unstained (R1); viable
cycling cells, >2n DNA content and merocyanine
540-unstained (R2); viable resting cells undergoing apoptosis,
2n DNA content and merocyanine 540-stained (R3); viable
cycling cells undergoing apoptosis, >2n DNA content and
merocyanine 540-stained (R4); and late stage apoptotic cells that are
merocyanine 540-stained but Hoechst 33342-unstained indicating DNA
fragmentation (R5). The data shown are representative of two
independent experiments. The untreated population contained merocyanine
540-stained cells owing to spontaneous apoptosis that occurred during
cell culture. The percent increase (mean ± S.D. from two
experiments) in apoptotic cells (i.e. merocyanine
540-stained cells in the R3, R4, and R5 compartments) in the irradiated
population over the untreated population was 6.18 ± 1.02 in
Egr-1
/
cells and 52.97 ± 2.32 in Egr-1+/
cells.
/
Cells--
To ascertain whether radiation
up-regulates p53 mRNA and the p53 target genes such as
p21waf1/cip1, mdm-2, and bax, we
performed 32P-RT-PCR using the RNA extracted from untreated
and irradiated cultures at various time points. In
Egr-1+/
cells, p53 mRNA was elevated after
15-30 min of irradiation (Fig. 3). Both
p21waf1/cip1 and mdm-2 were elevated in 30 min (Fig.
3); bax was also elevated up to 1 h of exposure to
ionizing radiation in Egr-1+/
cells (Fig. 3).
However, in Egr-1
/
cells, no
up-regulation was evident for p53, p21waf1/cip1,
mdm-2, and bax genes (Fig. 3). The basal levels
of p53, p21waf1/cip1, mdm-2, and
bax were higher in
Egr-1
/
cells as compared with
Egr-1+/
cells. Recently, it was reported that
EGR-1 protein directly binds to Rel homology domain in p65 (Rel A)
subunit of NF
B complex (39), and p65 was found to transactivate the
p53 promoter (40, 41). High basal levels of p53 and its
target genes in Egr-1
/
MEFs
(Fig. 3) may be attributed to induction of p53 promoter by
elevated NF
B activity in the absence of Egr-1 function.
Thus, absence of induction of these genes after radiation may have
contributed to enhanced radiation resistance in
Egr-1
/
cells.
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Fig. 3.
RT-PCR analysis of p53,
p21waf1/cip1, mdm-2, and bax
in untreated or irradiated Egr-1+/
or Egr-1
/
MEF cells.
Egr-1+/
or
Egr-1
/
MEF cells were left
untreated (UT) or treated with radiation (5 Gy), and total
RNA was extracted at the indicated time points. PCR was performed by
using the products of reverse transcription reaction and the upstream
and downstream primers flanking the p53,
p21waf1/cip1, mdm-2, and bax genes (Table
I) and
-actin gene as an internal control. bp, base
pair.
/
MEF Cells--
Western blot analysis was
performed to examine whether exposure to ionizing radiation caused
induction of p53 protein. Egr-1+/
and
Egr-1
/
cells were either left
untreated or exposed to a 5-Gy dose of ionizing radiation, and proteins
were extracted at various time intervals and subjected to Western blot
analysis for p53 protein. As seen in Fig.
4, a strong induction of p53 protein was
noticed in Egr-1+/
cells after irradiation;
after 3-6 h of radiation, p53 protein levels were increased about
5-fold in Egr-1+/
cells. However, in
Egr-1
/
cells, the p53 protein
was down-regulated after 1 h of radiation and reduced to <10% of
basal levels at 6- and 12-h time points (Fig. 4). The above
observations have ascertained the fact that EGR-1 protein is necessary
to cause radiation-induced apoptosis. Absence of EGR-1 protein renders
enhanced resistance to radiation. Thus, the loss of p53 protein in
Egr-1
/
cells after radiation may
have contributed to enhanced resistance to apoptosis.
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Fig. 4.
p53 protein is down-regulated by ionizing
radiation in Egr-1 /
cells.
Protein levels of p53 were detected by Western blot analysis before and
after radiation. Whole cell protein extracts were prepared from
Egr-1+/
and
Egr-1
/
cells that were left
untreated (UT) or exposed to a 5-Gy dose and incubated for
the time interval indicated and then subjected to Western blot analysis
for p53 or
-actin.
/
Cells Led to
Restoration of Sensitivity to Radiation-induced Apoptosis--
To
understand the regulation of p53 by Egr-1, we transiently
transfected Egr-1
/
cells by
using CMV-EGR-1 or vector-alone constructs. Transiently transfected
cells were left untreated and irradiated, and then either total
proteins were extracted for Western blot analysis of p53 protein or
TUNEL was performed at 24 or 48 h after irradiation. In
Egr-1
/
cells transfected with
vector alone, p53 levels were down-regulated after radiation (Fig.
5A), and these cells showed 8 or 11% cell death at 24 or 48 h after radiation, respectively
(Fig. 5B). On the other hand,
Egr-1
/
cells transfected with
CMV-EGR-1 showed no down-regulation of p53 protein (Fig. 5A)
and 10 or 38% cell death at 24 or 48 h after radiation,
respectively (Fig. 5B).
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Fig. 5.
Restoration of radiation sensitivity and
stabilization of p53 protein in Egr-1 /
MEF cells transiently transfected with CMV-EGR-1. A,
protein levels of p53 were detected by Western blot analysis before and
after radiation. Whole cell protein extracts were prepared from
vector-transfected Egr-1
/
cells
or CMV-EGR-1-transfected Egr-1
/
cells that were left untreated (UT) or exposed to a 5-Gy
dose and incubated for the time interval indicated and then subjected
to Western blot analysis for p53 or
-actin. B,
radiation-induced apoptosis in
Egr-1
/
transfectants was
determined by TUNEL assay. Transfectants were left untreated
(UT) or irradiated at 5-Gy dose of radiation and subjected
to TUNEL analysis. Approximately 1000 cells in total were scored for
TUNEL-positive cells in each experiment. The data shown here are the
percent TUNEL-positive cells as a function of irradiation.
C, absence of Egr-1-mediated transactivation in
transiently transfected p53-CAT reporter construct in irradiated
Egr-1
/
MEF cells.
Egr-1+/
and
Egr-1
/
cells were transiently
transfected with 4 µg of p53-CAT reporter plasmid. Transfected cells
were left untreated or irradiated at 5 Gy, and CAT activity was assayed
and normalized by determining the percent conversion of
[14C]chloramphenicol to acetylated forms using
densitometric ratios.
/
cells. Basal and
irradiated p53-CAT reporter activity was examined in
Egr-1+/
and
Egr-1
/
MEF cells. Both MEF cells
showed basal p53-CAT activity; however, higher CAT activity was
observed in Egr-1+/
cells than
Egr-1
/
cells (Fig.
5C). Radiation elevated the p53-CAT activity in
Egr-1+/
cells, whereas radiation caused no
change in the p53-CAT activity in
Egr-1
/
cells (Fig.
5C). These results indicate that Egr-1 may
transregulate p53. Together, Egr-1 is pivotal to mediate the
apoptotic action by ionizing radiation.
/
MEF Cells Led
to Modest Induction of Radiation-induced Apoptosis--
Our data in
Fig. 5C indicated lack of p53 promoter activity after
radiation in Egr-1
/
cells, and
this prompted us to understand precisely the cooperative role of
Egr-1 and p53 in the regulation of radiation-induced
apoptosis. We transiently transfected p53+/+ and
p53
/
MEF cells by using CMV-EGR-1 or
CMV-WT1-EGR-1 (dominant-negative mutant of EGR-1) or vector
pCB6+ constructs. Transiently transfected cells were left
untreated and irradiated, and then TUNEL was performed 24 and 48 h
after radiation (Fig. 6).
View larger version (22K):
[in a new window]
Fig. 6.
Radiation-induced apoptosis depends on both
functionally active EGR-1 and p53 proteins. Radiation-induced
apoptosis was quantified by TUNEL assay using vector or CMV-EGR-1 or
CMV-WT1-EGR-1-transfected p53+/+ (A) and
p53 /
(B) MEF cells. Transfectant
cells were left untreated (UT) or irradiated at 5-Gy dose of
radiation and after 24 and 48 h were subjected to TUNEL analysis.
Approximately 1000 cells in total were scored for TUNEL-positive cells
in each experiment. Data shown are percent TUNEL-positive cells as a
function of irradiation dose. Data represents a mean of three
experiments. The error bars represent S.D.
/
MEF cells caused modest induction of
cell death after 48 h of radiation when compared with
p53
/
MEF cells overexpressing vector alone
(Fig. 6B). Overexpression of dominant-negative mutant EGR-1
in p53
/
MEF cells showed reduction in
radiation-induced cell death when compared with cells overexpressing
vector alone. These data strongly suggest that both functional EGR-1
and p53 are essential to mediate radiation-induced apoptosis; however,
absence of p53 may not contribute toward complete abrogation of
EGR-1-mediated radiation-induced apoptosis.
/
Cells--
Recently, it was reported (30) that Rb regulates the
stability and the apoptotic function of p53 via MDM2 (42). It is also
known that EGR-1 regulates Rb through its consensus site on the
Rb promoter. Since Rb critically regulates the stability of
p53 protein, we hypothesized that the degradation of p53 protein after
radiation in Egr-1
/
cells may
due to loss of Egr-1-mediated transactivation of
Rb gene. To test this hypothesis, we performed the following
experiments to understand the mechanism of p53 degradation in
irradiated Egr-1
/
cells.
and
Egr-1
/
MEF cells. Western blot
analysis showed low levels of Rb including hypophosphorylated forms in
Egr-1
/
MEF cells before and
after radiation when compared with Egr-1+/
cells (Fig. 7A). Rb-CAT
reporter assay also indicated low basal CAT activity in
Egr-1
/
cells when compared with
Egr-1+/
cells (Fig. 7B). After
radiation, Rb-CAT activity was elevated to 2-fold in
Egr-1+/
cells but not in
Egr-1
/
cells (Fig.
7B). Recent studies have demonstrated that MDM2-p53 interaction directly targets p53 degradation (43). Since p53 was
degraded in irradiated Egr-1
/
cells, we performed Western blot analysis to determine the levels of
MDM2 in untreated and irradiated Egr-1+/
and
Egr-1
/
MEF cells. Interestingly,
MDM2 levels were down-regulated after radiation in
Egr-1
/
cells; on the other hand,
MDM2 levels were up-regulated after radiation in
Egr-1+/
cells (Fig. 7A).
View larger version (33K):
[in a new window]
Fig. 7.
Lack of hypophosphorylated form of Rb protein
leads to MDM2-mediated p53 degradation in
Egr-1 /
cells. A,
protein levels of Rb and MDM2 were detected by Western blot analysis
before and after radiation. Whole cell protein extracts were prepared
from Egr-1+/
and
Egr-1
/
cells that were left
untreated (UT) or exposed to a 5-Gy dose and then after
1 h of incubation were subjected to Western blot analysis for Rb,
MDM2, or
-actin. Expression of
-actin was used as loading
control. B, absence of Egr-1-mediated
transactivation in transiently transfected Rb-CAT reporter construct in
irradiated Egr-1
/
MEF cells.
Egr-1+/
and
Egr-1
/
cells were transiently
transfected with 4 µg of p53-CAT reporter plasmid. Transfected cells
were left untreated or irradiated at 5 Gy, and CAT activity was assayed
and normalized by determining the percent conversion of
[14C]chloramphenicol to acetylated forms using
densitometric ratios. C, Rb forms a complex with p53 through
MDM2. Cell lysates were prepared from Egr-1+/
and Egr-1
/
cells that were left
untreated (UT) or exposed to a 5-Gy dose, and after 1 h
of incubation were subjected to immunoprecipitation. The bound
complexes of Rb, p53, and MDM2 was detected by coprecipitating Rb with
MDM2 antibody and coprecipitating MDM2 with p53 antibody and subjected
to Western blot analysis.
/
cells when compared with
Egr-1+/
cells. Based on these observations, we
hypothesized that p53 degradation in irradiated
Egr-1
/
cells might be due to the
presence of higher amounts of p53-MDM2-bound forms and relatively lower
amounts of Rb bound to the p53-MDM2 complex (trimeric complex of
Rb-MDM2-p53). To test this hypothesis, we performed immunoprecipitation
experiments followed by Western blot analysis with cell lysates from
untreated and irradiated Egr-1+/
and
Egr-1
/
MEF cells. Radiation
caused high levels of Rb-MDM2 complex relative to p53-MDM2 complex in
Egr-1+/
cells (Fig. 7C). By
contrast, higher amounts of p53-MDM2 complex and lower amounts of
Rb-MDM-2 complex were observed in
Egr-1
/
cells after radiation
(Fig. 7C). Thus, the degradation of p53 in
Egr-1
/
cells after radiation may
be due to diminished Rb binding to MDM2 and enhanced MDM2 binding to
p53. Because of diminished Rb binding to MDM2, p53 is directly degraded
by MDM2, and thus the p53-mediated apoptotic pathway in
Egr-1
/
MEF cells is inactivated.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
transactivation (36). These results suggest that
Egr-1 induction is involved in the radiation-induced
signaling of the cascades of apoptosis pathway.
MEF
cells, Egr-1
/
MEF cells were
significantly resistant to radiation-inducible apoptosis and showed no
elevation of p53 protein after radiation. These observations indicate
that radiation-induced EGR-1-mediated transactivation of downstream
genes is essential for radiation sensitivity. Thus, in support of
previous reports, the present study demonstrates that EGR-1 is the
upstream mediator for the initiation of the radiation-induced signaling
cascade leading to cell death.
cells but not in
Egr-1
/
cells. In addition, the
basal levels of these mRNAs were high in
Egr-1
/
MEF cells when compared
with Egr-1+/
cells. Loss of radiation-induced
elevation of p53 may be attributed to the loss of Egr-1
mediated transregulation of p53 in
Egr-1
/
MEF cells, and this may
have led to the loss of up-regulation of p53 target genes,
p21waf1/cip1, mdm-2, and bax.
/
cells, and this led to
enhanced resistance to radiation-inducible apoptosis. Transient
overexpression of EGR-1 protein in
Egr-1
/
cells restored radiation
sensitivity and stabilized the p53 protein levels. Thus, this
observation suggests that EGR-1 protein is necessary for the
up-regulation and the stability of p53 protein and radiation
sensitivity. Moreover, radiation elevated the p53-CAT reporter activity
in Egr-1+/
cells but not in
Egr-1
/
cells. This observation
is supported by a recent study that EGR-1 can directly bind with the
p53 promoter at two consensus EGR-1-binding sites and induce
the p53 mRNA and protein (32). Thus, Egr-1 is an
important transregulator of p53. However, at this point we cannot rule
out the possibility that other genes that are also regulated by
Egr-1 may play a role in the stability of p53 protein.
/
/CMV-EGR-1 MEF transfectants when
compared with p53+/+/CMV-EGR-1 MEF cells suggests that p53
played an important downstream role in regulation of
Egr-1-mediated radiation-induced apoptosis. It also suggests
that the absence of p53 may not contribute toward complete abrogation
of EGR-1-mediated radiation-induced apoptosis. This is supported by our
previous data that in p53 null prostate cancer cell line PC3, EGR-1
overexpression caused super induction of radiosensitivity (36). The
degree of induction of apoptosis was much higher in p53 null PC3 cells
when compared with p53
/
/CMV-EGR-1 MEF
transfectant cells in this study. The difference may be due to the
tumor cell background versus the normal cell background.
Thus, in the absence of p53, EGR-1 may mediate the proapoptotic action
of radiation via TNF-
(36) or other downstream cell-death effector genes.
/
MEF cells, we investigated the expression and functional interaction of
Rb with p53 and MDM2 in Egr-1+/
and
Egr-1
/
MEF cells. The rationale
for analyzing the Rb function in this normal isogenic cell system is
that (a) Rb regulates the apoptotic function of p53 by
mitigating MDM2 mediated degradation (42) and (b) the Rb
gene promoter contains EGR-1-binding sites that conform to the GC-rich
consensus (30). Low expression levels of hypophosphorylated forms of Rb
and decreased Rb-CAT reporter activity were found in
Egr-1
/
MEF cells before and
after irradiation when compared with Egr-1+/
MEF cells. Relatively higher levels of Rb-MDM2-bound complex and lower
levels of p53-MDM2-bound complex were observed in irradiated Egr-1+/
MEF cells. In contrast, higher amounts
of p53-MDM2 complex and low bound forms of the Rb-MDM2 complex were
observed in Egr-1
/
cells. Lower
amounts of the Rb-MDM2 complex along with higher amounts of p53-MDM2 in
Egr-1
/
MEF cells might have
contributed to p53 degradation after radiation. Thus, apoptosis caused
by ionizing radiation requires the induction of EGR-1 protein, which
then transregulates the expression of p53 protein and also indirectly
regulates the stability of p53 via Rb.
![]() |
ACKNOWLEDGEMENTS |
---|
We thank Dr. Tyler Jacks for providing
primary cell culture of p53+/+ and
p53/
mouse embryonic fibroblasts and Dr.
Moshe Oren for providing p53-CAT constructs.
![]() |
FOOTNOTES |
---|
* This work was supported by United States Army Medical Research Grant DAMD17-98-1-8473 (to M. M. A) and in part by the Charlotte Geyer Foundation (to M. M. A).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.
§ Both authors contributed equally to this work.
** To whom correspondence should be addressed: Dept. of Radiation Medicine, University of Kentucky, 800 Rose St., Lexington, KY 40536. Tel.: 859-323-6904 (ext: 1021); Fax: 859-257-7483; E-mail: ahmm@pop.uky.edu.
Published, JBC Papers in Press, October 16, 2000, DOI 10.1074/jbc.M008454200
![]() |
ABBREVIATIONS |
---|
The abbreviations used are:
EGR, early growth
response;
Egr-1, early growth response-1 gene, CAT,
chloramphenicol acetyltransferase, EBS, EGR-1-binding site, MEF, mouse
embryonic fibroblast;
Gy, gray;
CMV, cytomegalovirus;
PCR, polymerase
chain reaction;
RT-PCR, reverse transcriptase-PCR;
TUNEL, terminal
transferase-mediated dUTP-digoxigenin nick end labeling;
TNF-, tumor
necrosis factor-
;
PDGF, platelet-derived growth factor;
Rb, retinoblastoma gene.
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