From The Burnham Institute, La Jolla Cancer Research Center, La
Jolla, California 92037, the § Sidney Kimmel Cancer
Center, San Diego, California 92121, the Cancer Center, Universtiy of
California at San Diego, La Jolla, California 92093, and the
¶ Istituto di Ricovero e Cura a Carattere Scientifico,
Neuromed, Pozzilli 86077, Italy
Received for publication, October 8, 2002, and in revised form, January 28, 2003
In the majority of aggressive tumorigenic
prostate cancer cells, the transcription factor Egr1 is overexpressed.
We provide new insights of Egr1 involvement in proliferation and
survival of TRAMP C2 prostate cancer cells by the identification
of several new target genes controlling growth, cell cycle progression,
and apoptosis such as cyclin D2, P19ink4d, and Fas. Egr1 regulation of
these genes, identified by Affymetrix microarray, was confirmed by
real-time PCR, immunoblot, and chromatin immunoprecipitation assays.
Furthermore we also showed that Egr1 is responsible for cyclin D2
overexpression in tumorigenic DU145 human prostate cells. The
regulation of these genes by Egr1 was demonstrated using Egr1 antisense
oligonucleotides that further implicated Egr1 in resistance to
apoptotic signals. One mechanism was illustrated by the ability of Egr1
to inhibit CD95 (Fas/Apo) expression, leading to insensitivity to FasL.
The results provide a mechanistic basis for the oncogenic role of Egr1
in TRAMP C2 prostate cancer cells.
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INTRODUCTION |
Prostate cancer is the most common malignancy in men and a
frequent cause of cancer death. The mortality of this disease is due to
metastasis to the bone and lymph nodes. Prostate cancer progression is
thought to proceed from multiple defined steps through prostatic
intra-epithelial neoplasia, invasive cancer, and progression to
androgen-independent and refractory terminal phase (44, 50). A large
fraction of early onset, and up to 5-10% of all prostate cancer
patients, may have an inherited germline mutation that has facilitated
the onset of carcinogenesis. However, in the majority of cases, no
inherited gene defects are involved, and cancer arises as a result of a
series of acquired somatic genetic changes affecting many genes on
several chromosomes. Although the molecular mechanism of prostate
cancer progression remains largely unknown, a few genes such as
E-cadherin,
-catenin,
TGF-
,1 and insulin-like
growth factors I and II have been shown to be aberrantly expressed and
are markers of prostate cancer (34, 69). To clearly understand the
multistep progression of this disease many other genes remain to be identified.
One of the overexpressed genes found in prostate cancer tissue is the
transcription factor early growth response gene 1 (Egr1) (18, 62). This
gene could have an important function because its expression level
increases with the degree of malignancy as measured by the Gleason
grade of the tumor (18). This seems to be specific to prostate tumor
cells, because in mammary and lung tumors, as well as most normal
tissues, Egr1 expression is low. Egr1 overexpression is correlated with
the loss of its co-repressor NAB2 in primary prostate carcinoma. This
disruption of the balance between Egr1 and NAB2 expression results in a
high Egr1 transcriptional activity in prostate carcinoma cells (1). A
recent study based on the cross breeding of Egr1
/
mice
with TRAMP mice showed significantly delayed prostate tumor formation
in the Egr1-deficient TRAMP mouse compared with
TRAMP-Egr1+/+ mice (2). The TRAMP mouse is a well known
model of prostate cancer (20) in which tumors progress to metastases in
a window from 8 to 24 weeks of age. Although Egr1 loss did not appear
to prevent tumor initiation, Egr1 deficiency delayed the progression of
prostate tumors in these mice. Significantly, several gene products
associated with aggressive prostate cancer such as TGF-
and
insulin-like growth factor II (37, 60) have been identified as
regulated by Egr1. These observations strongly suggest that Egr1 is
involved in prostate cancer progression despite its known role as a
tumor-suppressor in several other types of human cancers (29).
In this present study on the role of Egr1, we have used the tumorigenic
C2 prostate cancer cell line which was established from a prostate
tumor from a single TRAMP mouse tumor. These tumorigenic cells express
a high constitutive level of Egr1 protein. Transcriptional regulation
by Egr1 was assessed using Affymetrix array technology. The unique step
used here was to perform a microarray analysis using cells rendered
deficient in Egr1 as the comparison sample for the identification of
Egr1 target genes, in prostate cancer cells. The results provide new
insight into the involvement of endogenous Egr1 in proliferation and
survival of prostate cancer cells by the identification of several new
target genes specifically controlling growth, cell cycle progression,
and the apoptosis pathway.
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MATERIALS AND METHODS |
Cell Culture and Transfection Condition--
C2 TRAMP cells were
grown as described elsewhere (20). The cells were seeded into 35-mm
dishes at a density of 100,000 cells per well 1 day before
transfection. The transfection was performed as described by the
manufacturer with the GenePorter reagent (16 µl) (Gene Therapy
Systems, Inc, San Diego, CA) and 0.1 µM antisense oligonucleotide (AS or ctl). Sequences of the AS and mismatch control
oligonucleotide (ctl) were used as described (65). The sequence of ctl
oligonucleotide corresponds to AS sequence with 4 bases mutated.
Proliferation Assay and Cell Death Measurement--
One day
before transfection the cells were seeded in duplicate into 35-mm
dishes at a density of 70,000 cells per dish. At day 0 cells were
transfected as described above. 4 h later the cells were harvested
for counting and for protein and total mRNA extraction. This
procedure was repeated each day after transfection according to a time
course from day 0 to day 6.
The day after transfection, the cells were ultraviolet-C (UVC)
irradiated (40 J/m2) in a Stratalinker (Stratagene, La
Jolla, CA) or treated with 100 ng/ml of Fas L recombinant protein
(Oncogene Research Products, Darmstadt, Germany). One or two days after
UVC irradiation or 9 and 18 h after Fas L treatment, detached and
trypsinized cells were pooled and incubated with 0.2% trypan blue to
determine the percentage of dead cells.
Colony Forming Assay--
C2 cells were transfected as described
above. After 16 h the cells were counted and seeded into 6 well
plates (200 cells/well) in RPMI medium with 0.1 µM of
antisense oligonucleotide. After 8 days incubation at 37 °C, the
colonies were stained with 2% crystal violet.
Oligonucleotide Microarray Analysis--
The protocol
recommended by Affymetrix (www.affymetrix.com) was used for
mRNA quality control and gene expression analysis from C2 cells
transfected either with AS or ctl oligonucleotides. The probes were
hybridized to Affymetrix MGU75Av2 arrays representing ~12,000 mouse
transcripts. Detailed protocols for data analysis and documentation of
the sensitivity, reproducibility, and other aspects of the quantitative
microarray analysis using Affymetrix technology were used as reported
previously (39).
Quantitative Real-time One-step RT-PCR and Western
Blot--
mRNA expression level was quantified by real-time
one-step RT-PCR using the LightCycler-RNA-Amplification Kit SYBR Green
I (Roche Molecular Biochemicals) according to the
manufacturer's instructions. A standard curve from several dilutions
of a sample of total RNA was established to calculate the relative
amount of each gene. Values were then normalized to the relative
amounts of glyceraldehyde-3-phosphate dehydrogenase determined from a similar standard curve. Each gene was amplified using the appropriate specific primers (sequences available upon request).
For the Western blot analysis, proteins were blocked and reacted with
antibodies to Egr1 (C19, Santa Cruz Biotechnology, Inc., Santa Cruz,
CA), mouse and human cyclin D2 (sc-593 and sc-181, Santa Cruz
Biotechnology, Inc.), p19ink4d (sc-1063, Santa Cruz
Biotechnology) or CD95 (anti-mouse Fas/TNFRSF6 (CD95) antibody, R & D
Systems, Inc., Minneapolis, MN).
Chromatin Immunoprecipitation Assay--
To cross-link protein
on DNA targets the cells were incubated in 1% formaldehyde at 4 °C
during 30 min. After extraction as described elsewhere (13), the
chromatin was fragmented by sonication to obtain an average size of
1.5-kb DNA fragment. The DNA fragments mix was then immunoprecipitated
using a specific Egr1 antibody and a non-immune serum as a
negative control. After cross-link reversal as described elsewhere
(13), the screening for identification of the regulatory sequence of
captured Egr1 target genes was performed by PCR using the following
primers located in the 5' regulatory sequences of the following genes:
p19ink4d, 5'-ctggtcgctgcacgctgac-3' (forward) and
5'-agtggataccggtggactgt-3' (reverse) (
599 and
1, respectively, from
the ATG); cyclin D2, 5'-ggcgagctgaggagagccg-3' (forward) and
5'-ctccatagccagccggcca-3' (reverse) (
269 and +6, respectively, from
the ATG); cyclin G2, 5'-ccagcatcccccaagctact-3' (forward) and
5'-cttcatctgcagcaaatacacc-3' (reverse) (
601 and +6, respectively,
from the ATG); Mad, 5'-aagcggccggtggcccgc-3' (forward) and
5'-gctgtcgccatcctgcacc-3' (reverse) (-48 and +11, respectively, from
the ATG); CD95, 5'-cagtggtgagtcagtgggttt-3' (forward) and
5'-gacagcccagatccacagcat-3' (reverse) (
272 and +345, respectively,
from the ATG).
Genomic DNA input was used as a control for the amplification
efficiency of each primers pair. Non-immune immunoprecipitated DNA and
DNA immunoprecipitated from AS-transfected C2 cells were used as
negative controls. The amplified products were resolved on 2.7%
agarose gel.
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RESULTS AND DISCUSSION |
AS Antisense Oligodeoxynucleotide Efficiently Inhibits Egr1
Expression--
To examine the functional significance of Egr1
overexpression in prostate cancer cells, we inhibited its expression
using an AS in TRAMP C2 prostate cancer cells. To assess the efficiency and the specificity of AS, we performed Western blot analyses of the
protein expression of Egr1 and other Egr family members, Egr2, Egr3,
and wt1, 24 h after transfection of the antisense and control
oligonucleotides (Fig. 1A). As
seen in Fig. 1A, the antisense oligonucleotide strongly
decreased Egr1 expression, while there was no effect on Egr2 and WT1
expression. Egr3 seems to be slightly increased when Egr1 was
inhibited. In contrast, the ctl did not alter the protein expression
pattern of the cells. These results demonstrated that a 24-h treatment
with a low concentration, 0.1 µM, of the AS
oligonucleotide efficiently and specifically inhibited Egr1 expression.
To examine the time course of Egr1 inhibition in C2 cells, proteins
were extracted each day for 6 days following AS transfection of
antisense and control oligonucleotide-treated cells. Egr1 expression in
the presence of AS was undetectable from day 1 to day 3, became
detectable on day 4, and was fully restored on day 5 to day 6 (Fig.
1B, top panel). As expected, the use of the ctl
did not change Egr1 expression level (Fig. 1B, bottom
panel). These results show that AS is stable enough over 3 days to
allow almost complete and specific inhibition of Egr1 expression for a
prolonged period following a single treatment.

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Fig. 1.
Inhibition of Egr1 expression by E5 antisense
oligonucleotide. A, C2 cells were transfected with the ctl,
the AS, or carrier alone (M) for 4 h. After 24 h
the cells were lysed, and samples were analyzed by Western blotting
with antibodies to Egr1. Membranes were reprobed successively with
antibodies to Egr-3, Egr-2, WT-1, and -actin as internal control.
B, proteins were extracted every day for 6 days following AS
(C2-AS) and ctl (C2-ctl) transfection. Samples
were analyzed by Western blotting with antibody to Egr1 and antibody to
-actin to control for protein loading.
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Egr1 Contributes to the Control of Proliferation--
To determine
the involvement of Egr1 in the proliferation rate of C2 cells, the
growth of the cells in which Egr1 expression was inhibited by AS
oligonucleotide (C2-AS) was compared with the control corresponding to
C2 cells transfected with control oligonucleotide (C2-ctl). Briefly the
cells were transfected at day 0 with either AS or ctl and the
proliferation rate was directly assessed every day until day 6 by cell
counting (Fig. 2A). As seen in
Fig. 2A, the proliferation rate of C2-AS cells was strongly reduced during the first 3 days after transfection and started to rise
again on day 4. Between day 4 and 5 the slope of the proliferation curve was approximately equal to the slope of the control (C2-ctl cells), indicating that the cells recovered their expected
proliferation rate (Fig. 2A). The proliferation time course
was well correlated to the pattern of Egr1 inhibition seen in Fig.
1B. Indeed, as long as Egr1 expression was inhibited, the
proliferation rate of C2 cells was markedly reduced and then resumed as
soon as Egr1 expression recovered. In addition, comparison between
C2-AS and C2-ctl cells in a colony forming assay showed 74% fewer
colonies in C2-AS (average of 32 colonies (±6) for C2-ctl
versus 8.3 colonies (±3) for C2-AS), suggesting that
the tumorigenicity of the cells may decrease when Egr1 is inhibited
(Fig. 2B). Furthermore, cell cycle analysis by
fluorescence-activated cell sorter, performed at day 2 after
transfection, showed fewer cells (about 11% less) in the
G1 phase of C2-ctl cells than C2-AS cells (data not shown). The sum of results strongly argue in favor of a role for Egr1 in the
control of growth and cell cycle progression in prostate cancer
cells.

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Fig. 2.
Effect of Egr1 inhibition on
proliferation. A, proliferation assay. C2 cells were
transfected with ctl (C2-ctl) or AS (C2-AS)
antisense oligonucleotide and submitted to proliferation assay for 7 days. Each day from day 0 (D0) to day 6 (D6), the number of cells of
C2-ctl (solid line) and C2-AS (dashed line) was
counted and plotted as the mean of three separate experiments.
B, colony forming assay. C2 cells were transfected with 0.1 µM ctl or AS antisense oligonucleotide for 4 h.
After 16 h, 200 cells were placed in each well of six-well plates
in RPMI medium containing 0.1 µM antisense
oligonucleotides. After 8 days, the colonies were stained with 2%
crystal violet.
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Identification of Egr1 Target Genes by Affymetrix Microarray
Hybridization--
To determine the genes that are involved in
Egr1-mediated transformation, comparative analyses of mRNA
populations from C2 cells 1 day after transfection with AS or with ctl
oligonucleotide were performed using Affymetrix microarray
hybridization. Affymetrix analysis revealed a large number of genes (at
least 180) involved in the control of proliferation, death, and
malignant progression. Most of these had not previously been identified
as part of an Egr1 signaling pathway. Although many genes are direct
Egr1 target genes, others could be indirectly regulated by
Egr1 or modulated after the change of the physiological behavior
of the cells due to the inhibition of Egr1 expression. However
it is important to consider that those genes could be as important as
the direct target genes to maintain, potentiate, or regulate
Egr1 effect. Genes displaying the highest Affymetrix expression
changes upon treatment with AS are listed in Table
I.
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Table I
Affymetrix analysis of genes regulated in C2 cells that express Egr1
constitutively compared with antisense treated cells
For each gene, the -fold induction (Affymetrix ratio), its function
(gene function), any reported involvement in human prostate cancer
(link with prostate cancer), and data on its regulation by Egr1 (known
as Egr1 target gene) are given. TPA,
12-O-tetradecanoylphorbol-13-acetate.
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To confirm the Affymetrix microarray analysis results, the expression
of some genes listed in Table I was independently tested by
quantitative real-time RT-PCR. In these experiments, total RNA extracts
from Egr1 expressing and non-expressing C2 cells were used as
templates. The -fold induction/repression calculated from real-time
RT-PCR assays compared with the corresponding ratio determined in the
Affymetrix analysis (Table II), produced
remarkable concordance. The induction or repression of specific target
genes by Egr1 was in the same direction in all cases examined and
commonly exhibited a similar degree of change. Indeed, the Pearson
correlation coefficient of the Affymetrix and real-time PCR results was
0.78, which is significant (p = 0.008,
2). These results confirm the reliability of the
Affymetrix analysis.
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Table II
Comparison of Affymetrix array with real-time RT-PCR ratio for mRNA
levels
Changes in the expression level of several Egr1 target genes given in
Table I were independently tested using quantitative RT-PCR analysis of
RNA from C2-ctl and C2-AS treated cells. The results were normalized to
glyceraldehyde-3-phosphate dehydrogenase and expressed as the ratio of
C2-AS over C2-ctl values. All reactions were performed in triplicate
from two different experiments, and the resulting S.E. values are also
given. Positive and negative values mean, respectively, up-regulation
and down-regulation in response to Egr1 inhibition (positive values
indicate a down-regulation by Egr1).
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Examination of Table I reveals several candidate genes already
identified as Egr1 targets, such as transforming growth factor beta 1 (37, 60) and CD95 (15). Expression of other genes such as transcription
factor LRG-21/Atf3 and cyclin D2 is known to be correlated with an
increase of Egr1 expression (22, 35). Furthermore, several genes
identified here, have been directly linked to human prostate cancer.
Indeed, inhibitor
B
(I
B
) was shown in several prostate
cancer cell lines to inhibit growth, angiogenesis, and metastasis by
inhibition of NF-
B activity (31). Mad, by interacting with Max, is
known to prevent the transforming effect of Myc by inhibition of the
Myc/Max association (8). In addition, Myc is often found to be
overexpressed in prostate cancer cells (48). Accordingly, Egr1 could
promote Myc-induced transformation by down-regulation of Mad
expression. Apolipoprotein D secretion is associated with
steroid-induced inhibition of cell proliferation in the LNCaP human
prostate cancer cell line (59). Expression of this protein is low in
prostate cancer cells and can be modulated by steroid hormones and
other factors involved in the control of cell proliferation (59).
Caspase 7 and CD95 (Fas/APO) are known to be involved in the apoptotic
response in various prostate cancer cell lines (9, 40). Interestingly all these genes, which behave as tumor suppressors, are down-regulated by Egr1 in C2 prostate cancer cells. On the other hand, genes like
IGFBP-4, which stimulates cell proliferation in ALVA31 and M12 human
tumor prostate cells (16), and TGF-
1, which is strongly expressed in
prostate cancer cells (71, 72), are up-regulated by Egr1.
In summary, the genes that are involved in cell cycle progression,
malignant transformation, or inhibition of apoptosis are all
up-regulated by Egr1, while those involved in growth inhibition and
apoptosis are repressed (Table I). Hence constitutive expression of
Egr1 in prostate cancer affects the balance between survival and tumor suppression.
Characterization of Egr1 Regulation--
As seen in Fig.
1B, efficient inhibition of Egr1 expression occurred for 3 days after AS transfection. Thus, a similar time course of expression
should be expected for Egr1 target genes. Therefore, mRNA
expression of cyclin D2 and G
12 protein, both known to
stimulate growth and cell cycle progression (4, 68), p19ink4d
and cyclin G2, which inhibit cell cycle progression (26, 27), were
measured daily from day 0 to day 6 by real-time quantitative RT-PCR.
Cyclin D2 and G
12 mRNA expression was drastically
inhibited from day 1 to day 3 when Egr1 was inhibited and resumed as
soon as Egr1 expression was normal (days 4-6) (Fig.
3). Similarly, synthesis of cyclin G2 and
p19ink4d mRNAs was increased until day 3 when Egr1
expression was low (Fig. 3), and normal expression was restored on day
4. These results were not observed upon treatment with the ctl
oligonucleotide, demonstrating that Egr1 expression is absolutely
required for full mRNA expression of cyclin D2 and
G
12 and to repress p19ink4d and cyclin G2
mRNA synthesis. The duration of this regulation (at least 3 days)
demonstrates that no other transcription factor compensates for the
lack of Egr1 function in these cells.

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Fig. 3.
Time course of mRNA expression.
Cyclin D2, G 12 protein, cyclin G2, and p19ink4d
mRNA expression were determined by one-step real-time RT-PCR.
Expression levels of each gene were normalized to the level of
glyceraldehyde-3-phosphate dehydrogenase expression and the ratio
between each day versus day 0 was calculated as -fold
induction. All reactions were performed in duplicate from two different
samples corresponding to C2 cells transfected with ctl (black
columns) or AS (gray columns) antisense
oligonucleotide.
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To test whether mRNA regulation mediated by Egr1 is reflected at
the protein level, immunoblotting analysis was performed on proteins
extracted from C2-AS and C2-ctl cells from day 0 to day 6 after
transfection. In these experiments cyclin D2 and p19ink4d
protein expression level was assessed. The results showed a
time-dependent repression of cyclin D2 and an increase of
p19ink4d protein expression (days 1-3) (Fig.
4A) in antisense treated cells, which matched the time course of their mRNA expression patterns (Fig. 3). These results confirm that Egr1 inhibition by
antisense is efficient enough to modulate Egr1 target gene expression
at the protein level. In addition, the cyclin D2 and p19ink4d
time-dependent protein expression patterns (from day 0 to
day 6) are also highly correlated to the difference found in the cell cycle analysis and in the proliferation rate (Fig. 2A)
between C2-AS and C2-ctl cells. This finding corresponds to their
activities in the regulation of cell cycle progression. Thus cyclin Ds
are required for cell cycle progression and overexpression of INK4 family proteins is responsible for the G1 phase arrest (54, 55). Interestingly, cyclin D2 is found to be up-regulated by Egr1,
while p19ink4d expression, a cyclin D2-dependent
kinase inhibitor (5), is repressed. Therefore Egr1, by reciprocally
regulating the levels of p19ink4d and cyclin D2, would
stimulate cell cycle progression and play a prosurvival role in
prostate cancer cells.

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Fig. 4.
Time course of protein expression.
A, time course regulation of cyclin D2 and p19ink4d
protein expression. Protein extracts from C2-ctl and C2-AS cells were
analyzed as described in Fig. 1, A and B, by
Western blotting with antibodies to Egr1, cyclin D2, p19ink4d
using -actin as a loading control. B, cyclin D2,
G 12 protein, and p19ink4d mRNA expression
were determined by one-step real-time RT-PCR in human prostate DU145
cells. Expression levels of each gene were normalized to the level of
glyceraldehyde-3-phosphate dehydrogenase expression, and the ratio
between AS condition versus ctl oligonucleotide was
calculated as -fold induction. C, protein extracts from
DU145 (lanes 1, 2, 3, and
6), 267B (lane 4), P69 (lane 5) cells
were analyzed by Western blotting with antibodies to Egr1, cyclin D2,
and -actin.
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To test the generality of these results, we examined p19ink4d,
cyclin D2, and G
12 protein expression by real-time
RT-PCR in the human prostate cancer cell line, DU145, transfected
either with AS or ctl oligonucleotide. As in C2 cells, Egr1 expression
is constitutively high in DU145 and strongly inhibited by the antisense oligonucleotide (Fig. 4C, left panel). In DU145,
Egr1 regulation of these genes appeared to be the same as the
regulation observed in the C2 mouse model (Fig. 4B).
Furthermore cyclin D2 protein expression is also strongly repressed
during the inhibition of Egr1 expression, indicating that Egr1 is
required to maintain cyclin D2 protein expression level in DU145 as
well as in mouse TRAMP C2 cells (Fig. 4C, left
panel). To examine Egr1 and cyclin D2 expression during human
prostate cancer progression, we tested three additional cell lines,
normal 267B1 prostate epithelial cells, low tumorigenic P69 cells, and
aggressively tumorigenic DU145 human prostate cells. While Egr1
expression is similar in normal human prostate 267B and P69 cell lines,
it is overexpressed in DU145. Thus cyclin D2 expression correlates with
Egr1 expression in these cell lines and is strongly expressed only in
the aggressive tumorigenic DU145 cells (Fig. 4C, right
panel). These results support the relevance of C2 cells as a model
to identify new Egr1 target genes in prostate cancer.
Egr1 Desensitizes the Cells to Fas L-induced Apoptosis--
Egr1
may also play a role in promoting prostate cancer by affecting prostate
cell survival (30) or apoptosis (65), and this was tested next. C2-AS
and C2-ctl cells were UVC-irradiated, and dead cells were counted by
trypan blue staining 24 and 48 h later. While less than 20% of
the C2-ctl cells were dead 24 h following irradiation, almost 50%
of C2-AS cells were dead (Fig. 5A). Furthermore, at 48 h
following irradiation, less than 50% of control cells
versus 95% for C2-AS cells had died (Fig. 5A). These differences demonstrate a critical role for Egr1 in response to
stress. Indeed, endogenous expression of Egr1 is not only required for
full proliferation of C2 cells but also to decrease sensitivity to
radiation, a widely observed phenomenon of human prostate cancer cells
(28).

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Fig. 5.
Inhibition of Egr1 expression increases
sensitivity to apoptotic stimuli. A, C2-ctl and C2-AS
were exposed or not to UVC radiation (40 J/m2). One and two
days later dead cells were determined by trypan blue staining. The blue
staining dead cell count is shown as a percentage of the total cells,
and the absolute number of dead and alive cells is reported within the
bar chart. B, C2-ctl and C2-AS were exposed (lanes
2 and 4) or not (lanes 1 and 3)
to UVC radiation (40 J/m2). Twenty-four hours later
proteins were extracted and subjected to analysis by Western blotting
with antibodies to Egr1 or CD95. -Actin level were used as a loading
control. C, Fas L-mediated apoptosis. C2-ctl and C2-AS were
treated or untreated with 100 ng/ml Fas L for 9 and 18 h as
described under "Materials and Methods." Dead cells were
determined by trypan blue staining and reported as described
above.
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Affymetrix analysis (Table I) revealed several genes that are
down-regulated by Egr1, such as caspase 7 (6, 40), Bcl-2-binding protein homolog Nip3 (10) and CD95 (Fas antigen) (9), a gene widely
involved in apoptosis pathways. CD95, a member of tumor necrosis factor
receptor family, is referred as "death receptor" because of its
ability to transduce death signals. On the other hand, the gene
PS-2short (up-regulated by Egr1, see Table I) is involved in inhibition
of Fas-mediated apoptosis (66, 67), therefore supporting a role for
Egr1 as anti-apoptotic agent in prostate cancer cells.
Egr1 regulation of CD95, although confirmed at the mRNA level by
real-time PCR (Table II), was also tested for protein expression in
C2-ctl and C2-AS cells treated or not by UVC irradiation. In C2-ctl
cells, UVC treatment led to a significant increase of Egr1 expression,
which was strongly inhibited by AS (Fig. 5B,
C2-AS). CD95 expression appeared to be undetectable in
C2-ctl-treated cells but was clearly expressed in C2-AS-treated cells
(Fig. 5B). After UVC treatment CD95 expression was strongly
increased in C2-AS, while it was only slightly expressed in C2-ctl
(Fig. 5B). These results confirm at the protein level the
efficient inhibition of CD95 expression by Egr1. This mechanism of
repression is all the more relevant, since it is still effective even
after a strong stress stimulus.
To assess whether this difference in basal CD95 expression could be
reflected as responses to Fas L mediated apoptosis, we treated C2-ctl
and C2-AS cells for 9 and 18 h with Fas L and counted the
percentage of dead cells by trypan blue staining. As expected from CD95
protein expression profile (Fig. 5B), C2-AS were more sensitive to Fas L-mediated apoptosis. Indeed, at 9 h after
treatment, 52% of cells were dead in C2-AS versus 18.5% in
C2-ctl cell cultures (Fig. 5C). This difference in the
resistance to cell death between C2-AS and C2-ctl cells, although
lower, was still present after 18 h treatment, with 96% of dead cells
compared with 60%, respectively (Fig. 5C). Therefore high
constitutive Egr1 expression delays apoptosis of prostate cancer cells
mediated by Fas L, in part by down-regulating CD95 expression. The
significance of the CD95 signaling pathway in prostate apoptosis has
also been demonstrated in the normal rat prostate following castration
(14). In addition, further studies have demonstrated the involvement of
CD95 in sensitizing prostate cancer cells to undergo apoptosis after
chemotherapeutic agent or irradiation treatments (12, 33). These
results illustrate well a "desensitizer role" of Egr1 in the cell
death response and suggest that sensitization to Fas-mediated
apoptosis by the inhibition of Egr1 expression could become an
attractive therapeutic mechanism. Furthermore this experiment presents
corroborating evidence that the modification of gene expression by Egr1
is a major player in the pathological responses of prostate cancer cells.
p19ink4d, Mad, CD95, and Cyclin D2 Are Directly
Transcriptionally Regulated by Egr1--
Gene chip and real-time PCR
technologies are powerful and sensitive enough to accurately evaluate
the differential expression between two mRNA populations, but do
not determine whether the regulation by Egr1 occurs directly or
indirectly. Therefore, we performed chromatin cross-linking and
immunoprecipitation assays (ChIP) to screen upstream regulatory
sequences of five examples of putative Egr1 target genes indicated by
the Affymetrix analysis. For this experiment untransfected, AS and ctl
oligonucleotide-transfected C2 cells were used. After chromatin
cross-linking in living cells, Egr1 became covalently fixed to its DNA
target. These captured target DNA fragments were then recovered by
specific Egr1 immunoprecipitation and purification. Non-immune serum
immunoprecipitation was used as the negative control and C2 genomic DNA
was used to assess amplification efficiency of each primer pair.
Primers were designed to specifically recognize 5' regulatory sequences
of p19ink4d, Mad, CD95, cyclin G2, and cyclin D2, to detect
their presence in the captured DNA fragments by polymerase chain
reaction. 5' regulatory sequence analysis of each of these genes showed
several putative Egr1 and Sp-1 binding sites. p19ink4d, Mad,
CD95, and cyclin D2 yielded an amplified product from untransfected (Mock) (Fig. 6A)
and ctl oligonucleotide-transfected template (Fig. 6B) that
showed the same migration pattern as the genomic control input, while
cyclin G2 was not detected (Fig. 6, A and B).
Since no amplification was found for the control non-immune serum
template (Fig. 6A) and the AS oligonucleotide-transfected template (Fig. 6B), these results indicate that the
successfully amplified fragments were bound by Egr1 in vivo
and therefore indicate the direct regulation of p19ink4d, Mad,
CD95, and cyclin D2 by Egr1. Furthermore, to rule out the possibility
that these genes could be regulated in consequence of the inhibition of
the proliferation, we performed a kinetic study of the regulation of
TGF-
1, a well known Egr1 target gene (73). Since AS
oligonucleotide is effective at 5 h after transfection (data not
shown), we performed the kinetic analysis at 5, 10, and 15 h. As
for TGF-
1, the modulation of cyclin D2 and p19ink4d
expression occurred at 5 h after AS addition corresponding to the
onset of Egr1 efficient inhibition (Fig. 6C). Taken
together these results indicate that many of the Egr1 target genes
identified in our study may be regulated directly by Egr1.

View larger version (50K):
[in this window]
[in a new window]
|
Fig. 6.
Egr1 binds directly to p19ink4d, Mad,
CD95, and cyclin D2 regulatory sequences. C2 cells were
transfected (B) or not (A) with AS and ctl
oligonucleotides. The cells were chromatin cross-linked and then
immunoprecipitated with specific Egr1 antibody or nonimmune control
antibody. The detection of each gene in the captured fragment mix, was
performed by PCR as described under "Materials and Methods."
A, the top, middle, and bottom
panels show, respectively, PCR products from the genomic DNA
input, Egr1-specific immunoprecipitation samples, and the non-immune
control from untransfected C2 cells (Mock). B,
the top and bottom panels show, respectively, PCR
products from Egr1-specific immunoprecipitation samples from C2
transfected with ctl and AS oligonucleotides. C, cyclin D2,
p19ink4d, and TGF- 1 mRNA expression were determined by
one-step real-time RT-PCR. Expression levels of each gene were
normalized to the level of glyceraldehyde-3-phosphate dehydrogenase
expression, and the ratio between 5, 10, and 15 h
versus 0 h was calculated as -fold induction.
|
|
 |
CONCLUSIONS |
Our study provides new insight on the activities and mechanisms of
Egr1 in prostate cancer cells. We propose that Egr1 promotes cell
growth and desensitization to death by regulating a set of genes known
to be very important in cell cycle progression, growth, and apoptosis.
Therefore, constitutive Egr1 expression observed here in prostate
cancer cells is likely to promote both tumor cell growth and
progression. We suggest that our results extend the findings of
Milbrandt and co-workers (2) in that they indicate the mechanistic
basis of the role of Egr1 in cancer growth as well as progression. Our
study confirms for the first time in prostate, the growth enhancer role
of Egr1 previously observed in other cellular systems such vascular
smooth muscle and rat kidney tumor cells (19, 49). However, these roles
are tissue-specific, because in breast cancer, fibrosarcoma, and
glioblastoma, Egr1 behaves as a tumor suppressor gene (7, 29) that can
be required for maximal sensitivity to irradiation (3, 65). Further
comparisons of the identity and the regulation of Egr1 target genes
from these different tissues will explain this functional discrepancy.
We thank Dr. Nigel Mackman for helpful
comments on the work.
Published, JBC Papers in Press, January 29, 2003, DOI 10.1074/jbc.M210279200
The abbreviations used are:
TGF, transforming
growth factor;
AS, antisense oligonucleotide;
ctl, control
oligonucleotide;
RT, reverse transcriptase;
I
B
, inhibitor
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