1 Department of Medicine, University of Minnesota Medical School, Minneapolis,
MN 55455, USA
2 Department of Genetics, Cell Biology, and Development, University of Minnesota
Medical School, Minneapolis, MN 55455, USA
* Author for correspondence (e-mail: steer001{at}tc.umn.edu )
Accepted 13 May 2002
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Summary |
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Key words: Apoptosis, Cytokine, E2F transcription factors, Human hepatoma cells, TGF-ß1
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Introduction |
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It has been reported that overexpression of E2F-1 induces cells to undergo
apoptosis, and can occur by both p53-dependent and -independent mechanisms
(Holmberg et al., 1998). The
cooperation between E2F-1 and p53 may provide an apoptotic signal when it
occurs simultaneously with an arrest of cell cycle progression, such as by
p53. The ability of E2F-1 to induce apoptosis was thought to be a unique
feature compared with the other members of the E2F transcription family
(DeGregori et al., 1997
).
However, it has subsequently been determined that other members of the E2F
family (Harbour and Dean,
2000
), in particular E2F-3
(Ziebold et al., 2001
), make
major contributions to the apoptotic pathway. In fact, it has recently been
reported that Apaf-1, the gene for apoptosis protease-activating
factor 1, is a transcriptional target for both E2F and p53
(Moroni et al., 2001
).
TGF-ß1 is a potent growth inhibitor, and can induce rapid growth
arrest and apoptosis in many cell types, including hepatic cells in culture
and in vivo (Oberhammer et al.,
1992; Fan et al.,
1995
). Its growth suppressive effects appear to be linked, in
part, to decreased phosphorylation of pRb. Also, the inhibition of pRb
expression by TGF-ß1 in human HuH-7 hepatoma cells is associated with
significant apoptosis. The inactivation of pRb either by phosphorylation,
mutation, or binding to an oncoprotein results in the loss of its ability to
sequester E2F-1 as a pRbE2F-1 complex, and an increase in unbound E2F-1
(Riley et al., 1994
;
Fan and Steer, 1996
). E2F-1 is
the best-characterized member of the E2F transcription factor family
(Kaelin et al., 1992
;
Shan et al., 1996
), and its
overexpression is sufficient to offset pRb-mediated G1 arrest and
thereby promote S phase entry (Johnson et
al., 1994
). This is presumed to occur through transactivation of
the E2F-1 target genes dihydrofolate reductase (DHFR)
(Blake and Azizkhan, 1989
),
thymidylate synthase, and other factors involved in entry and progression
through S phase (Ishida et al.,
2001
; Müller et al.,
2001
).
It is now well established that apoptosis is a gene-directed process and
involves an intrinsic, albeit complex, cell death program that is regulated by
the cell cycle (Kroemer et al.,
1995). In fact, cell cycle progression and programmed cell death
appear to share a number of common pathways, as well as specific factors
(Evan et al., 1995
). For
example, pRb is an important cell cycle regulator and functions to inhibit
cell proliferation by complexing with transcription factor E2F at the
G1/S check-point. However, it is also known that the loss of
functional pRb can induce apoptosis while its expression can preserve cell
survival (Haas-Kogan et al.,
1995
; Fan and Steer,
1996
). Interestingly, TGF-ß1-induced apoptosis was preceded
by cell cycle arrest in HuH-7 cells. In fact, 93% of the preapoptotic cells
were initially arrested in G1 and eventually died from apoptosis
with continued exposure to TGF-ß1
(Fan et al., 1996
). In
contrast, deregulated expression of E2F-1 can drive quiescent cells into S
phase, ultimately resulting in apoptosis
(Qin et al., 1994
;
Shan and Lee, 1994
). The
mechanism by which TGF-ß1 induces apoptosis through deregulation of pRb
expression and phosphorylation in HuH-7 cells may, in fact, involve a number
of factors, although E2F-1 appears to be a key regulator in this pathway. In
fact, the abundance and accessibility of E2F-1 is critical to the regulation
of the cell cycle by pRb. Untimely activation of E2F-1 and/or its
overexpression may act to initiate TGF-ß1-induced apoptosis.
In this study, we report that E2F-DNA-binding activity is significantly increased in TGF-ß1-induced apoptosis of HuH-7 cells, in part, through the loss of interaction with pRb. The increased levels of unbound E2F family members may result in transactivation of several well-characterized cell-cycle-regulated genes. While the role of E2F in apoptosis is closely linked to its interaction with pRb, it functions in a complex array of pathways that occur in both the G1 and S phases of the cell cycle. The preapoptotic changes are partially reversible only upon removal of TGF-ß1, and can be inhibited by overexpression of pRb. The E2F family members are key regulators of cell survival and cell death.
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Materials and Methods |
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For G1 phase synchronization, cells were starved in DMEM medium containing 0.1% FBS for 48 hours, then replaced with isoleucine free medium (Atlanta Biologicals) containing 5 µM each of deoxycytidine, deoxyadenosine, deoxyguanosine and deoxythymidine (Sigma, St Louis, MO) for 30 hours. The efficiency of synchronization was monitored by flow cytometry. For S- and G2/M phase, the medium was removed after G1 phase synchronization and the cells were washed twice with 1x PBS, pH 7.4. The cells were incubated with fresh DMEM containing 10% FBS plus aphidicolin (2.5 µg/ml) for 18 hours, and then washed twice with 1x PBS. For S phase synchronization, fresh DMEM medium supplemented with 10% FBS was added and the cells incubated for an additional 2 hours before harvesting. For G2/M phase, the cells were incubated in DMEM supplemented with 10% FBS and 80 ng/ml of nocodazol for 18 hours, with the addition of 0.06 µg/ml colcemid for the final 2 hours prior to harvesting.
Flow cytometry
HuH-7 cells were harvested after either TGF-ß1 incubation or
synchronization, and analyzed by flow cytometry as described previously
(Fan et al., 1996). In brief,
the culture medium was collected and combined with the PBS washes. The
attached cells were removed with trypsin and were centrifuged at 500
g for 10 minutes at 4°C. The resulting pellets
(
5x105 cells) were resuspended in 0.5 ml of ice-cold 70%
ethanol and maintained on ice for at least 30 minutes. The cells were then
centrifuged and resuspended in 0.5 ml of PBS containing 0.1 mg/ml RNase A
(Roche Diagnostics, Indianapolis, IN), 50 µg/ml propidium iodide (Sigma),
and 0.05% (v/v) Triton X-100 (Sigma), and incubated for 45 minutes at room
temperature. The samples were analyzed using a FACS Star Plus sorter
(Becton-Dickinson, San Jose, CA) with 200 mW argon laser excitation at 488 nm.
Cell clumps were identified and gated out by plotting integral red
fluorescence versus peak height. The cell cycle distribution was established
by plotting the intensity of the propidium iodine signal, which reflects the
cellular DNA content. Apoptotic cells were identified as a hypodiploid DNA
peak representing cells that contained less than a 2N DNA content.
Preparation of nuclear extracts
All reagents were from Sigma unless otherwise indicated. The cells were
lysed with hypotonic buffer (10 mM Hepes pH 7.9, 1.5 mM MgCl2, 10
mM KCl, 0.2 mM PMSF, 0.5 mM DTT, 0.5 mM NaF, 0.5 mM
Na3VO4) on ice for 20 minutes and dounce homogenized
using ten strokes with the B pestle. The lysate was centrifuged at 3300
g for 15 minutes at 4°C, the supernantant discarded and
the nuclear pellet resuspended in 1.66 volumes of extraction buffer (20 mM
Hepes, pH 7.6, 1.6 M KCl, 20% glycerol, 0.1 mM EDTA, 0.5 mM NaF, 0.5 mM
Na3VO4, 0.5 mM PMSF, 1 mM DTT, 1 mg/ml leupeptin and
aprotinin) and gently stirred at 4°C for 30 minutes. The sample was
centrifuged at 32,000 g for 1 hour at 4°C, and the
supernatant dialyzed for 1 hour at 4°C against 20 mM Hepes, pH 7.6,
containing 50 mM KCl, 10% glycerol, 0.1 mM EDTA, 1 mM DTT, 0.1 mM PMSF, 0.1 mM
NaF, 0.1 mM Na3VO4 plus 1 mg/ml leupeptin and aprotinin.
Protein concentrations of the dialyzed nuclear extracts were determined by the
Bradford method using a commercial protein assay system (Bio-Rad Laboratories,
Hercules, CA). The extracts were then aliquoted, flash-frozen in liquid
nitrogen and stored at -70°C.
Gel-shift assays
E2F electrophoretic mobility shift assays were performed as described
previously (Cao et al., 1992).
The E2F-specific (GAT TTA AGT TTC GCG CCC TTT CTC AA) or mutant
(GAT TTA AGT TTC GAT CCC TTT CTC AA) synthetic oligonucleotides
(Integrated DNA Technologies, Coralville, IA) were labeled with
[
-32P]dCTP. Mobility shift assays were performed in 20 µl
reaction mixtures containing 20 µg of nuclear extract in 20 mM Hepes, pH
7.6 with 40 mM KCl, 1 mM MgCl2, 0.1 mM EGTA, 0.4 mM DTT, 5 µg
BSA, 2.5% (v/v) Ficoll, 2 µg of salmon sperm DNA and 32P-labeled
probe at 20,000-50,000 cpm. `Supershift' experiments were performed by
incubating extracts with 2 µg of the relevant antibodies for 10 minutes on
ice. These included anti-pRb XZ161 generously provided by Ed Harlow (Harvard
Medical School, Boston, MA), anti-p107, anti-p130, anti-DP-1, anti-DP-2,
anti-E2F-1, anti-E2F-2, anti-E2F-3 (Santa Cruz Biotechnology, Santa Cruz, CA)
and anti-MDM2 (Oncogene Research Products, Boston, MA). For binding
specificity, 10-, 100- and 1000-fold excess of unlabeled E2F-specific
oligonucleotides were coincubated with the 32P-labeled probes. To
disrupt protein-protein interactions within the E2F complexes, nuclear
extracts were incubated with increasing concentrations of sodium deoxycholate
(DOC) (0.2%, 0.4%, 0.8%, 1.2%; v/v) for 20 minutes on ice and then NP-40 was
added to a final concentration of 0.7%
(Bagchi et al., 1990
). The
mobility shift assay reactions were incubated at room temperature for 30
minutes, the samples were then loaded on a 0.25x Tris-Borate-EDTA, 4%
polyacrylamide gel (30:1) and electrophoresed at 200 V for 2 hours. Gels were
dried and analyzed by autoradiography. The relative intensities of the bands
were determined using a BioRad model GS-700 imaging densitometer (Bio-Rad
Laboratories).
Transfections and CAT assays
Transfections were performed as previously described
(Fan et al., 1996). In brief,
5 µg of each construct was transfected into HuH-7 cells for 24 hours using
LipofectinTM (Life Technologies) according to the manufacturer's
recommendations. The reporter construct E2F1CAT consisted of the entire human
E2F-1 promoter fused to the chloramphenicol acetyltransferase (CAT) gene
(Johnson et al., 1993
);
4XE2FCAT was constructed with a synthetic promoter containing four E2F
consensus binding sites (Ohtani and
Nevins, 1994
) driving CAT expression. Cells were co-transfected
with a pRb expression construct pCMV.RB containing a CMV promoter
(Fan et al., 1996
). A lacZ
ß-galactosidase reporter construct was used as a control plasmid. The
cells were then incubated for an additional 36 hours with or without 1 nM
TGF-ß1. After removing dead cells by gentle washing, the remaining viable
cells were harvested for preparation of cell lysates for CAT ELISA assays
(Roche Diagnostics), which were performed according to the manufacturer's
specifications. All CAT activity was normalized to the observed
ß-galactosidase expression, thus controlling for differences in
transfection efficiency and nonspecific effects of cell culture.
Northern and western blot analysis
Total RNA was prepared from the cells, electrophoresed and transferred to
MSI MagnaGraph nylon membranes (Micron Separations, Westboro, MA) as
previously described (Johnson et al.,
1993; Fan et al.,
1996
). Lane loading was determined by ethidium bromide staining
and densitometric analysis as described previously
(Correa-Rotter et al., 1992
).
Northern blots were probed with a 0.7 kb EcoRI/BglII E2F-1
cDNA fragment that was labeled and detected using the DIG/GeniusTM System
(Roche Diagnostics) according to the manufacturer's protocol. Whole cell
lysates were prepared and analyzed by western blotting as described previously
(Fan et al., 1996
) using
monoclonal anti-E2F-1 antibody (Santa Cruz Biotechnology).
Immunohistochemistry
The cells were fixed with 4% paraformaldehyde (w/v) in 1x PBS, pH 7.4
at room temperature for 10 minutes, washed three times with 1x PBS, and
then stained with Hoechst dye 33258 (1 µg/ml) for 5 minutes and mounted
using an anti-fade reagent. The images were acquired and the nuclear
morphology was analyzed as described previously
(Fan et al., 1996).
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Results |
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Nuclear extracts were prepared from synchronized G1-, S- and
G2/M phase HuH-7 cells or TGF-ß1-induced preapoptotic cells.
Gel retardation assays of E2FDNA-binding complexes indicated at least
six different components in synchronized and preapoptotic G1
nuclear extracts (Fig. 2). The
same four E2FDNA-binding complexes were present in all samples prepared
with the extracts from the normal synchronized cells, while two novel
complexes (I and IV) were observed using extracts from the preapoptotic cells
(Fig. 2, lanes 1-4). Most of
the E2F-binding activity from the normal G1 cells was detected in
complexes II and III (Fig. 2,
lane 1). In contrast, the majority of increased E2F activity in the
TGF-ß1-induced preapoptotic G1 cells was associated with
complexes I, IV, V and VI (Fig.
2, lane 2). The total E2F-binding activity in S phase (measured as
band intensities) was greater than either the G1- or
G2/M phases of the synchronized normal cells
(Fig. 2, lanes 1,3,4). However,
much higher E2F activity was observed in TGF-ß1-induced G1
preapoptotic cells than in the synchronized G1-, S- and
G2/M-phase normal cells (Fig.
2, lanes 1-4). Interestingly, dramatic increases in E2F complexes
V and VI relative to G1 phase were also detected in reactions using
S phase nuclear extracts.
|
To determine the specificity of E2F binding, incubations were done with 10-, 100-, and 1000-fold excess of unlabeled wild-type or mutant E2F oligonucleotides. The results indicated that a 1000-fold excess of unlabeled wild-type oligonucleotide could effectively compete for the E2F-binding activity in either of the G1 nuclear extracts. In contrast, the mutant E2F oligonucleotide showed no effect (Fig. 2, lanes 5-12). Interestingly, the wild-type E2F oligonucleotides resulted in the rapid loss of complexes I to IV in both G1 synchronized populations, while the 1000-fold excess was required to diminish complexes V and VI.
Increased E2F activity results from unbound E2F and not changes in
transcript or protein expression
We determined whether the increased E2F-DNA binding activity observed in
both the synchronized S phase and TGF-ß1-treated preapoptotic cells
resulted from increased E2F-1 mRNA and/or protein steady-state levels. Both
northern and western blot analyses were performed on the same samples. In
fact, the results indicated no significant changes in the steady-state levels
of either the E2F-1 transcript or protein during the cell cycle
(Fig. 3A,B). Although the
expression of E2F-1 mRNA was minimally increased in TGF-ß1-induced
preapoptotic G1 cells (less than twofold), no significant change in
protein abundance was observed (Fig.
3A,B). However, we could not rule out changes in rates of de novo
synthesis versus degradation of E2F protein and/or transcript in the different
cell cycle phases.
|
E2Fprotein complexes are different in the two G1
cell populations
We detected at least six different E2F-DNA complexes in the nuclear
extracts of both synchronized and TGF-ß1-induced preapoptotic
G1 cells, with perhaps only two in common
(Fig. 4, lanes 1,6). These
results suggested that E2F cooperates with different cellular proteins in
G1 preapoptotic cells, permitting interactions with cell cycle
mediators distinct from those in synchronized G1 cells. To further
characterize these E2Fprotein complexes, we performed sodium
deoxycholate (DOC) dissociation assays
(Baeuerle and Baltimore, 1988).
DOC-induced loss of specific DNAprotein complexes results from
disruption of protein-protein interactions in complexes containing at least
two protein species (Bagchi et al.,
1990
). Thus, nuclear extracts from both synchronized and
preapoptotic G1 cells were incubated with DOC, resulting in a
concentration-dependent dissociation of complexes I to VI. While complexes I
to IV were completely dissociated with 0.8% DOC
(Fig. 4, lanes 4,9), complex V
was more sensitive and dissociated at 0.4% DOC. In contrast, complex VI was
relatively resistant to DOC, even at 1.2% concentration. The three novel gel
retarded bands that appeared after dissociation of the initial complexes
remained resistant to 1.2% DOC. Taken together, the results suggested that
each of the DNAnuclear extract complexes, derived from synchronized or
preapoptotic G1 cells, contains multiple and potentially distinct
cellular components.
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Super-shift gel retardation assays were then performed to identify partner
proteins in the E2F family of complexes
(Philpott and Friend, 1994).
pRb is a critical factor for E2F function and both its phosphorylation state
and expression are inhibited by TGF-ß1 in the preapoptotic cells
(Fan et al., 1996
). Therefore,
anti-pRb monoclonal antibody (XZ161) was used to detect pRb in complexes
derived from either the preapoptotic G1 or normal synchronized
G1 cells. Interestingly, anti-pRb antibodies produced a significant
shift only in complex VI of the four identified E2F-specific DNAprotein
complexes from the preapoptotic nuclei
(Fig. 5A, lanes 2,4). However,
when the antibody was added to the synchronized G1 nuclear
extracts, complexes VI, III and a significant percentage of complex II were
shifted (Fig. 5A, lanes 1,3).
The results suggested that complexes II and III contain pRb in normal
G1 and this interaction was lost in G1 preapoptotic
cells.
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Other pRb family members such as p107 and p130 are also known to interact
with members of the E2F family (Dyson,
1998). In fact, E2F-1, -2, and -3 can interact with pRb, but not
with p107 (Lees et al., 1993
).
E2F-4 and -5 both associate preferentially with p107 and p130, although each
can also interact with pRb (Moberg et al.,
1996
). Therefore, E2F-1, -2 and -3 as well as p107 and p130
antibodies were used in immuno-shift assays to investigate their presence in
complexes from both G1 cell cycle phases. Our results showed that
E2F-1, -2, and -3 are abundantly present in complexes III, IV and VI, and to a
minor extent in complex II. This suggests that complexes I, II and V contain
primarily E2F-4 and -5 (Fig.
5A, lanes 5-10). In contrast to pRb, the increased E2F activity
did not alter the pattern of E2F association with p107 or p130. Both p107 and
p130 were present in all the complexes that were derived from either the
preapoptotic or synchronized G1 cells
(Fig. 5B, lanes 7-10). However,
complex V shifted less dramatically in the preapoptotic cells
(Fig. 5B, lanes 2,8,10).
Moreover, p130 antibody completely shifted complexes I, II and VI and a
significant portion of complexes III and IV
(Fig. 5B, lanes 9,10). Taken
together, the data indicate that E2F-3 contributes significantly more than
E2F-1 and E2F-2 to the increased E2F-specific DNA-binding activity observed in
the preapoptotic G1 cells. Interestingly, p107 and p130 contributed
substantially to complexes IV and VI that were derived from preapoptotic
cells, which contained significant levels of E2F-1, -2 and -3.
It has been demonstrated that DP-1 and DP-2 associate with E2F-1, -2, and
-3 in vivo and the complexes can activate transcription of E2F-specific sites
(Bandara et al., 1994;
Krek et al., 1993
;
Wu et al., 1995
). Yet, such
heterodimers preferentially form complexes with pRb, but not p107 and p130,
which are the predominant pRb family members present in the E2F-DNA-binding
complexes (Hijmans et al.,
1995
; Sardet et al.,
1995
). Therefore, to characterize formation of E2FDP
complexes and thus their potential for transcriptional activition in
preapoptotic cells, anti-DP-1 and -DP-2 antibodies were used for super-shift
gel retardation analysis. As shown in Fig.
5B, DP-1 exhibits maximal association with E2F in synchronized
G1 cells but less so in preapoptotic cells (lanes 1-4). In both
G1 cell populations, DP-2 exhibited a similar capacity for complex
formation with some E2Fs, shifting an equivalent percentage of complexes I and
IV or II and III, respectively (Fig.
5B, lanes 1,2,5,6). In contrast, complex VI, although completely
shifted in synchronized G1 cells, showed only a slight decrease in
preapoptotic cells with anti-DP-2. Although total E2F-DNA binding activity is
significantly increased in preapoptotic cells, the extent of complex formation
with DP-1 and -2 was reduced.
In addition to interacting with the pRb and DP family members, E2F-1 can
bind to the oncogene MDM2, both in vitro and in vivo, and the formation of
such complexes appears to increase E2F-dependent transcription
(Martin et al., 1995). MDM2
can also physically interact with pRb and p53, inhibiting the pRb growth
regulatory function and p53-dependent transcription, as well as increasing the
degradation of p53 (Xiao et al.,
1995
). Thus, to determine whether the increase of unbound E2F in
preapoptotic cells results in increased levels of interaction between E2F and
MDM2, gel shifts were performed using anti-MDM2 antibodies. Our results
indicated that the MDM2 antibody shifted all the complexes more completely in
the synchronized G1 cells (Fig.
5B, lanes 1,11). In contrast, only complexes IV, V and VI derived
from the preapoptotic cells were partially shifted by the MDM2 antibody
(Fig. 5B, lanes 2,12). This
suggests that MDM2 from preapoptotic cells and synchronized G1
cells interact differently with E2F. However, the increased E2F activity in
preapoptotic G1 cells did not increase its level of association
with MDM2.
Increased E2F activity mediates TGF-ß1-induced apoptosis without
S phase entry
A number of studies have reported that ectopic expression of E2F-1 induces
rapid S phase entry and apoptosis (Shan
and Lee, 1994; Asano et al.,
1996
). Therefore, we determined whether the increased E2F activity
in TGF-ß1-induced preapoptotic cells was sufficient for S phase entry.
The cell cycle status of the remaining preapoptotic HuH-7 cells following 72
hours of TGF-ß1 incubation was assessed by flow cytometry. Our results
indicated that >90% of preapoptotic cells were in G1 phase
(Table 1). Thus, even though
similar dramatic increases in E2F-specific DNA-binding activity were observed
in the same two complexes (V and VI) in both S phase and TGF-ß1 treated
preapoptotic cells, S phase entry did not occur
(Fig. 2, lanes 1-3).
|
Removal of TGF-ß1 leads to release of preapoptotic cells from
G1 to S phase but does not completely inhibit apoptosis
To evaluate the potential irreversible apoptotic commitment induced by
TGF-ß1, the preapoptotic cells after 72 hours of exposure were released
into normal culture medium containing 10% FBS. These preapoptotic cells
rapidly entered S phase (47.9%) with some apoptosis (17%), resulting in a
dramatic decrease of the G1 cell population from 91% to 21% by 15
hours post-release (P<0.001)
(Table 1). G1 cells
increased from 21% at 15 hours to 27% at 24 hours post-release; meanwhile, S
phase cells increased slightly from 54% to 57% but with an additional 9% loss
of the preapoptotic cells to apoptosis. By 48 and 72 hours post-release, the
G1 cell population increased to 45%, and S phase cells decreased to
40%. Interestingly, the G2/M phase cell population was not
significantly different at any time post-release, reflecting a constant
7% of the total cells present by 24 hours post-release. In fact, after
the initial post-release loss,
7% of the remaining preapoptotic cells
died every 24 hours after removal of TGF-ß1. Taken together, the results
suggest that E2F-mediated S phase entry was dependent on the removal of
inhibitory signals but also the presence of appropriate cell cycle regulators
for entry into S phase. A significant percentage of the cells arrested in
G1 with TGF-ß1 were irrevocably committed to cell death, and a
return to normal cellular function could not be achieved even after removal of
the apoptotic stimulus.
Increased E2F activity in preapoptotic cells mediates transcription
that is reduced by overexpression of pRb
More than 1240 cellular genes have been identified that contain E2F sites
that contribute to transcriptional regulation
(Ishida et al., 2001;
Müller et al., 2001
).
Most studies have demonstrated that E2F is a positive mediator of
transcription (Flemington et al.,
1993
; Ginsberg et al.,
1994
). However, some results indicate that E2F may also involve
suppression of transcription (Johnson et
al., 1993
; Müller et al.,
2001
). Two CAT transcription reporter constructs were used to
determine whether the increased E2F-DNA binding activity observed in our
preapoptotic cell nuclear extracts could accelerate E2F-mediated
transcription. The E2F1CAT construct contained the entire human E2F-1 promoter
on a 1.5 kb ApaI fragment driving the CAT gene
(Johnson et al., 1993
). The
4XE2FCAT construct expressed CAT under control of an E2F-dependent test
promoter with E2F-specific DNA binding sites
(Ohtani and Nevins, 1994
).
HuH-7 cells were transiently transfected with either of the two different
reporter constructs and a ß-galactosidase-expressing reporter plasmid to
control for transfection and expression efficiency. After 24 hours, the
transfected cells were washed, and then incubated for 36 hours with either 1
nM TGF-ß1 or no addition. Prior to harvesting, the dead cells were
removed by gentle washing and the remaining attached cells harvested to
determine CAT activity. Treatment of the transfected cells with 1 nM
TGF-ß1 for 36 hours resulted in an increase of 84.8% in CAT reporter gene
expression with the E2FICAT and 138.5% with the 4XE2FCAT constructs
(Fig. 6A). In contrast, the
ß-galactosidase control plasmid showed equivalent levels of expression
irrespective of TGF-ß1 treatment (data not shown). This data, coupled
with the northern and western blot analyses of E2F-1 transcript and protein
levels, respectively, suggests that post-transcriptional mechanisms
predominately control the abundance of this E2F factor during TGF-ß1
induced apoptosis.
|
We previously demonstrated that overexpression of pRb prevented the
apoptotic cell death associated with increased expression of E2F-1 in HuH-7
cells (Fan et al., 1996).
Thus, co-transfection experiments were carried out using pCMV.RB to
overexpress pRb together with either the E2F1CAT or 4XE2FCAT and the
ß-galactosidase control plasmid. Post-transfection, the cells were
treated as outlined above and CAT activity assessed after 36 hours of
incubation in the presence or absence of 1 nM TGF-ß1. Overexpression of
pRb inhibited CAT expression from the co-transfected E2F1CAT or 4XE2FCAT by
48.7% and 36.7%, respectively (Fig.
6B). This suggests that some of the E2F-controlled transcriptional
activation induced by TGF-ß1 can be partially reduced by simultaneous
overexpression of pRb, confirming that the increased CAT expression results
from TGF-ß1-induced E2F activity in preapoptotic cells.
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Discussion |
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There is no compelling evidence that a transient S phase entry actually
exists prior to apoptosis. In fact, our previous studies of cell cycle
kinetics in asynchronous cell populations suggest that it does not.
TGF-ß1 arrested more than 90% of cells in the G1 phase and
continuous exposure to the cytokine resulted in apoptosis without cell cycle
progression (Fan et al.,
1996). When these preapoptotic G1 synchronized cells
were released from TGF-ß1 and placed in normal growth medium for 15
hours, S phase cells increased by 47.9%. However, 17% of the released
preapoptotic cells ultimately underwent apoptosis. The increase in these two
cell populations accounted for greater than 90% of the loss observed in the
G1 population. This implies that the loss of these preapoptotic
cells represents a portion of the TGF-ß1-treated cultures that were
irrevocably committed to apoptosis and did not recover even under favorable
growth conditions.
It is probable the apoptosis that occurred after removal of TGF-ß1 was
due primarily to cells already committed to cell death, as the characteristic
apoptosis-associated nuclear morphologic changes were observed microscopically
immediately after releasing the cells (data not shown). However, we cannot
rule out the possibility that the apoptosis occurs rapidly after S phase
entry. Several lines of evidence indicate that deregulated expression of
E2F-1, -2 and -3 does induce S phase entry and apoptosis
(Shan and Lee, 1994;
Vigo et al., 1999
). However,
our data suggest that TGF-ß1-induced apoptosis occurs predominately in
G1 phase. If we consider that this is a primary process, then
apoptosis induced by deregulated expression of E2F in S phase would be a
secondary process. In fact, the dramatically increased E2F-DNA binding
activity in the G1 preapoptoic cell population did not result in
progression to S phase, but rather apoptosis. Additionally, there were similar
increases in two E2F-specific DNA complexes relative to G1 phase in
both normal S phase and preapoptotic cells; yet two complexes, which were
identified in normal cells were absent and replaced by two unique complexes in
the preapoptotic cells. The differences in E2F-specific DNAprotein
complexes identified in nuclear extracts from S phase and preapoptotic cells
further suggest that the increased E2F activity had not induced S phase, but
rather a new apoptotic state.
The novel complexes identified in the preapoptotic state may, in part,
explain the lack of effective progression to S phase even in the presence of
greatly elevated E2FDNA-binding activity. For example, the unique
complexes I and IV are predominately E2Fs interacting with the two related pRb
family members, p107 and p130, which are now thought to be the primary
regulators of E2F-dependent cellular proliferation
(Hurford et al., 1997;
Takahashi et al., 2000
). In
fact, fibroblasts from pRb-deficient (pRb-/-) mouse embryos
exhibited little alteration in expression patterns of E2F target genes as well
as normal cell cycle regulation. Thus, p107 and p130 control the progression
of the cell cycle through E2F association in the absence of functional
pRb.
Alternatively, the increased nuclear E2F-DNA-binding activity observed may
be a contributing factor to the observed G1 arrest. Both E2F-4 and
-5 are actively exported from the nucleus during cell cycle entry, and this
export appears to be required for exiting G1
(Gaubatz et al., 2001).
Moreover, E2F-4- and E2F-5-deficient embryonic fibroblasts are unable to
undergo pocket protein-mediated G1 arrest although they proliferate
normally (Gaubatz et al.,
2000
). As the G1 arrest induced by pocket proteins is
dependent on their ability to form E2F-containing transcription-repression
complexes (Zhang et al.,
1999
), the data implies that E2F-4 and E2F-5 mediate pocket
protein-dependent transcriptional repression during G1. This
suggests that the unique complex I found in the preapototic cells comprised
predominately of E2F-4 and E2F-5, and their pocket protein partners p107 and
p130 prevent exit from G1 by maintaining transcriptional repression
of factors required for S phase entry. In fact, keratinocytes treated with
TGF-ß1 also accumulate an E2F-4/p130 complex that with histone
deacetylase HDAC1 directly represses cdc25A promoter activity,
compromising progression through G1, and thus promoting cell cycle
arrest in a quiescent state (Iavarone and
Massagué, 1999
).
E2F-mediated p53-dependent apoptosis has been extensively studied and
suggests that wild-type p53 and E2F-1 cooperate to induce apoptosis in
cultured cells (Wu and Levine,
1994). In transgenic pRb-knockout (pRb-/-) animals, the
massive apoptosis associated with increased levels of unbound E2F-1 resulting
in embryonic death is suppressed in certain tissues in fetuses from
pRb-/-/p53-/- mice
(Clarke et al., 1992
;
Morgenbesser et al., 1994
).
However, dual knockout pRb-/-/pE2F-1-/- mice indicate
that in the absence of E2F-1, although marked suppression of p53-dependent
apoptosis is observed, embryonic death resulting from apoptosis still occurs
(Tsai et al., 1998
). This data
suggests a significant role for other E2F family members in mediating this
process.
E2F-3 also appears to have a direct role in mediating apoptosis in the
absence of pRb, as shown in pRb/pE2F-3-deficient mice. In these animals, the
concurrent loss of E2F-3 activity prevented both p53-dependent and
-independent apoptosis observed in pRb-deficient animals
(Ziebold et al., 2001).
Interestingly, there was no reduction in cell number or evidence of apoptosis
in the fetal livers of the pRb/pE2F3-deficient mice in contrast to the
pRb-/- mice. The dramatic increase in E2F-1 and E2F-3 binding
activity coupled with the significantly decreased pRb levels in
TGF-ß1-treated cells suggests that the unique complexes identified in the
preapoptotic cells may also promote apoptosis via transcriptional
activation.
Increased E2F activity in TGF-ß1-induced preapoptotic cells was found
to increase E2F-related transcriptional activation. The increase of unbound
E2F in preapoptotic cells, due to the loss of pRb binding, can directly
activate expression of another set of apoptosis-related genes
(Sofer-Levi and Resnitzky,
1996; Müller et al.,
2001
). For example, c-Myc can induce apoptosis in different types
of cells (Evan et al., 1995
),
and its expression appears to be mediated, in part, by the transcription
factor E2F-1. In fact, the c-myc P2 promoter activation by E2F-1
induces efficient transcription of c-myc, and this transcriptional
activity can be abrogated by overexpression of pRb
(Oswald et al., 1994
).
Recently, microarray analysis has demonstrated that, in the absence of de novo
protein synthesis, the E2Fs directly induce the expression of several key
regulators of apoptosis including the effector caspases 3 and 7, as well as
the cytochrome-c-binding protein Apaf-1
(Müller et al., 2001
).
This suggests that the increased E2F activity observed in the preapoptotic
cells could directly induce apoptosis via transcriptional activation of
apoptosis-associated genes. However, this is most likely a secondary event in
TGF-ß1-induced apoptosis. Recently, we investigated the mechanism by
which the bile acid ursodeoxycholic acid inhibits TGF-ß1-induced
apoptosis in primary rat hepatocytes and HuH-7 cells
(Rodrigues et al., 1999
). We
determined that the earliest event induced by TGF-ß1 in both cell types
was a mitochondrial permeability transition (MPT). The MPT-induced
mitochondrial swelling results in the efflux of cytochrome c, which
clearly preceded and was required for the subsequent caspase activation,
poly(ADP-ribose) polymerase (PARP) cleavage and apoptotic nuclear morphologic
changes induced by TGF-ß1. This implies that the TGF-ß1-mediated
events involved in committing the cell to apoptoisis occur prior to the
potential transcriptional activation of apoptosis mediated by E2F.
Our results also demonstrated that the increased E2F transactivation was
inhibited by 50% with pRb overexpression, indicating that some but not
all of the increased activity was due to a loss of E2F sequestration by pRb.
This implies that another potential function that may be lost with inhibition
of pRb expression by TGF-ß1 is the ability of E2F to repress
transcription. In contrast to transcriptional activation by the E2Fs,
transcriptional repression requires de novo protein synthesis
(Müller et al., 2001
).
Thus, loss of pRb gene expression may result in the loss of transcription of
other protein products required to mediate this process. The altered
interactions of the E2Fs with DP-1 and -2 in the preapoptotic G1
versus normal G1 cells suggest a modulation in E2F transcriptional
targets. Alternatively, loss of pRb might affect this activity directly by
releasing active repression of proximally bound transcription factors
(Ross et al., 2001
), thereby
allowing expression of gene products that inhibit the repression by members of
the E2F family.
Therefore, we propose three steps or processes to describe the sequence of
events for TGF-ß1-induced apoptotsis in these cells. (1) TGF-ß1
inhibits phosphorylation of pRb and subsequently its expression, thereby
inducing apoptosis in HuH-7 cells. This is the primary apoptotic event and
appears to be mediated by MPT-induced mitochondrial swelling and rupture,
which results in an efflux of cytoychrome c, and is followed by
caspase activation, PARP cleavage and nuclear changes
(Rodrigues et al., 1999). (2)
The increased E2F activity is secondary to the inhibition of pRb expression
and yet does not lead to S phase entry or progression. This lack of cellular
proliferation is most likely due to the marked sequestration of the E2Fs by
p107 and p130 in the preapoptotic cells and lack of nuclear export of E2F-4
and -5. (3) The loss of sequestration of E2F by pRb permits unbound E2F to
activate the expression of apoptosis-related genes.
Thus, it appears that E2F-induced DNA synthesis and apoptosis may be
separable processes. In fact, apoptosis induced by E2F-1 may not depend on
E2F-1-dependent transcriptional activation
(Phillips et al., 1997;
Phillips et al., 1999
),
although DNA binding appears to be important in Saos-2 cells, which do not
express wild-type pRb or p53. Our studies showed that TGF-ß1 incubation
leads to significantly increased E2F-DNA-binding activity and stimulates
E2F-mediated transcriptional activation and apoptosis but not proliferation in
HuH-7 cells. It remains to be established whether the E2F-mediated
transcriptional activation is directly responsible for expression of the
cytochrome-c-binding protein Apaf-1 and effector caspases that are
activated subsequent to mitochondrial cytochrome c efflux.
Nevertheless, our studies indicate that overexpression of pRb can
significantly inhibit E2F-mediated transcriptional activation and apoptosis,
underscoring its critical role in regulating the life and death pathways
associated with E2F.
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