From the Department of Molecular Cell Biology, The Weizmann Institute of Science, Rehovot 76100, Israel
Received for publication, October 9, 2002, and in revised form, November 20, 2002
![]() |
ABSTRACT |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Exposure of cells to genotoxic agents results in
activation of checkpoint pathways leading to cell cycle arrest. These
arrest pathways allow repair of damaged DNA before its replication and segregation, thus preventing accumulation of mutations. The tumor suppressor retinoblastoma (RB) is required for the
G1/S checkpoint function. In addition, regulation of
the G2 checkpoint by the tumor suppressor p53 is
RB-dependent. However, the molecular mechanism underlying
the involvement of RB and its related proteins p107 and p130 in the
G2 checkpoint is not fully understood. We show here that
sustained G2/M arrest induced by the genotoxic agent doxorubicin is E2F-dependent and involves a decrease in
expression of two mitotic regulators, Stathmin and AIM-1. Abrogation of
E2F function by dominant negative E2F abolishes the doxorubicin-induced down-regulation of Stathmin and AIM-1 and leads to premature exit from
G2. Expression of the E7 papilloma virus protein, which
dissociates complexes containing E2F and RB family members, also
prevents the down-regulation of these mitotic genes and leads to
premature exit from G2 after genotoxic stress. Furthermore,
genotoxic stress increases the levels of nuclear E2F-4 and p130 as well
as their in vivo binding to the Stathmin promoter. Thus,
functional complexes containing E2F and RB family members appear to be
essential for repressing expression of critical mitotic regulators and
maintaining the G2/M checkpoint.
Cell cycle arrest in response to DNA damage is an important
mechanism for maintaining genomic integrity. This cell cycle arrest provides time for DNA repair to prevent replication or segregation of
damaged DNA. Induction of growth arrest by DNA damage occurs mainly through the activation of checkpoint pathways that delay cell cycle progression at G1, S, and G2
(1, 2).
Growth arrest at both G1 and G2 is believed to
occur in two steps, resulting in a rapid arrest that is followed by a
more sustained arrest. Initial arrest at G1 involves
phosphorylation and degradation of both the protein phosphatase cdc25A
and cyclin D1, resulting in inhibition of G1
cyclin-dependent kinases (3-5). Initial arrest at
G2 involves phosphorylation and inhibition of the protein
phosphatase cdc25C, leading to inhibition of cdc2 activity (6, 7).
Through transactivation of p21, the tumor suppressor p53 is one of the
essential mediators of sustained arrest at both G1 and
G2 in response to DNA damage (8-11). p21 binds to
cyclin-cyclin-dependent kinase complexes and
inhibits their ability to phosphorylate the retinoblastoma tumor
suppressor, RB,1 and its
related proteins, p107 and p130 (12). Cells deficient of RB fail to
arrest at G1 after DNA damage, indicating that RB plays an
essential role in this arrest (8, 13). In addition, p53 regulation of
DNA damage-induced G2 arrest was shown to be RB-dependent (14).
RB, p107, and p130 play a key role in negative regulation of cell cycle
progression, and their growth inhibitory activity is largely attributed
to their association with members of the E2F family of transcription
factors (15, 16). The E2F family is composed of six members,
E2F-1-E2F-6, which heterodimerize with the DP proteins, DP-1 or DP-2,
to form the DNA-binding, active transcription factor (15, 16). E2F
plays a crucial and well established role in the control of cell cycle
progression mainly by up-regulating expression of genes required for
the G1/S transition as well as for DNA replication (15,
16).
This transcriptional activity of E2F is inhibited by its interaction
with RB, p107, and p130 (15, 16). In addition, the complex containing
E2F and RB family members also actively represses transcription.
Assembly of such repressive complexes, containing E2F and RB family
members (referred to herein as E2F-RB, although they may contain p107
or p130), on promoters that have E2F-binding sites is critical for
growth suppression by RB family members (17, 18). The combination of
cessation of repression of some E2F-regulated genes by the E2F-RB
complex and the activation of others by activated E2F constitutes a
major step in promoting G1 exit.
E2F-1, -2, and -3 comprise a subgroup of the E2F family. These E2Fs are
specifically regulated by RB and not by the RB-related proteins, p107
and p130. Their release from RB precedes the activation of
E2F-responsive genes as well as S-phase entry (19), and their overexpression induces quiescent cells to enter S-phase (20-24). In
addition, E2F-1, and possibly also E2F-2 and -3, induce apoptosis (21,
24, 25). E2F-4 and -5 constitute another subgroup of the E2F family.
They interact also with p107 and p130 and are implicated mainly in
repression of gene expression (26, 27). Unlike E2F-1, -2, and -3, which
are constitutively nuclear, E2F-4 and -5 are found in the nucleus only
in G0 and early G1, when many of the
E2F-regulated genes are repressed (28-31). Furthermore, binding of
E2F-4 to promoters is associated with gene repression (26, 32).
Recent studies indicate that E2F regulates the expression of genes
involved not only in cell cycle progression but also in other
biological processes (33-36). Many of these novel E2F targets function
in various cellular responses to DNA damage, including activation of
checkpoints, DNA repair, and apoptosis, thus implicating E2F in the DNA
damage response. Further support to this notion comes from recent
reports demonstrating that the E2F-1 protein is stabilized and its
levels are increased following DNA damage (37-40). This stabilization
is due to phosphorylation of E2F-1 by the protein kinase ATM, one of
the master controllers of the response to DNA damage (41).
We and others have shown that E2F up-regulates expression of a number
of genes involved in entry to and progression through mitosis (33, 35).
However, the mode of regulation of these mitotic genes by E2F and the
biological consequences of this regulation are not fully understood. We
show here that expression of two of these mitotic genes, AIM-1 and
Stathmin, is also elevated by transcriptionally inactive E2F-1,
indicating that they are subjected to E2F-dependent
repression. Furthermore, we show that E2F-containing complexes are
required for DNA damage-induced down-regulation of AIM-1 and Stathmin.
This repression of gene expression is correlated with
E2F-dependent maintenance of DNA damage-induced growth
arrest at G2.
Cell Culture--
NIH3T3 cells were grown in Dulbecco's
modified Eagle's medium supplemented with 10% bovine calf serum.
Rat-1a-MT-wtE2F-1 and Rat-1a-MT-E2F-1dlTA, which are the Rat1 cell
lines transfected with an inducible plasmid expressing either wild type
of a dominant negative mutant of E2F-1, were grown in Dulbecco's
modified Eagle's medium supplemented with 10% fetal calf serum and
G418 (500 µg/ml). For zinc induction, cells were maintained
for 48 h in medium with 0.1% serum, and then 100 µM
ZnCl2 were added to the medium.
293 cells were grown in Dulbecco's modified Eagle's medium
supplemented with 10% fetal calf serum.
All cells were maintained at 37 °C in a humidified 8%
CO2- containing atmosphere.
Activation of the E2F-1 fused to estrogen receptor ligand-binding
domain (ER-E2F-1) was induced by addition of 4-hydroxytamoxifen to a
final concentration of 30 nM.
Cycloheximide was added to a final concentration of 10 µg/ml.
Doxorubicin was added to a final concentration of 0.2 µg/ml.
Plasmids--
The pSV- Retroviral Infection--
Cells of the packaging cell line 293 were co-transfected with 10 µg of RT-PCR--
Reverse transcription-PCR (RT-PCR) was performed on
total RNA prepared by the Tri Reagent method. For this assay, 7.5 µg
of RNA was employed for cDNA synthesis using Moloney murine
leukemia virus reverse transcriptase (Promega, 200 u) and
oligo(dT) (Amersham Biosciences, 0.5 µg). Following are the
number of cycles, annealing temperature, and the sequences of 5' and 3'
primers used for each of the tested genes, respectively: for the gene
encoding AIM-1, 28 cycles, 58 °C using 5'-AGATTGGGCGTCCTCTGGG and
5'-TCAATCATCTCTGGGGGCAG; for the gene encoding Stathmin, 35 cycles,
58 °C using 5'-GGTGAAAGAACTGGAGAAGCG and 5'-GTGCTTATCCTTCTCTCGC; for
the gene encoding ARPP-PO, 19 cycles, 58 °C using
5'-GTGGGAGCAGACAATGTGG and 5'-CAGCTGCACATCGCTCAGG; for the gene
encoding GAPDH, 20 cycles, 58 °C using 5'-ACCACAGTCCATGCCATCAC and
5'-TCCACCACCCTGTTGCTGTA.
Fractionation and Western Blotting--
For nuclear and
cytoplasmic fractions, cell pellets were resuspended in four packed
cell volumes of hypotonic buffer (10 mM HEPES, pH 7.9, 10 mM KCl, 1 mM EDTA, 1 mM
dithiothreitol, 1 mM phenylmethylsulfonyl fluoride,
20 µg/ml aprotinin, and 10 µg/ml leupeptin) and incubated for 15 min on ice. Cells were then lysed by adding Nonidet P-40 to a final
concentration of 0.6% and vortexing. After a short centrifugation, the
cytoplasmic supernatant was taken out, and the nuclear pellet was lysed
in two packed cell volumes of lysis buffer (20 mM HEPES, pH
7.9, 400 mM NaCl, 1 mM EDTA, 1 mM
dithiothreitol, 1 mM phenylmethylsulfonyl fluoride, 20 µg/ml aprotinin, and 10 µg/ml leupeptin). Protein
concentrations were determined by Bradford assay. The ratio of loaded
volumes of cytoplasmic and nuclear extracts from a given cell
population was equivalent to the ratio of volumes of hypotonic and
lysis buffers, respectively, and therefore it is considered as the per cell ratio of cytoplasmic and nuclear proteins. For whole cell extrcats, cells were lysed in lysis buffer (20 mM HEPES, pH
7.8, 450 mM NaCl, 25% glycerol, 0.2 mM EDTA,
0.5 mM dithiothreitol, 1 mM
phenylmethylsulfonyl fluoride, 20 µg/ml aprotinin, and 1 µg/ml leupeptin). Equal amounts of protein from each lysate,
as determined by Bradford assay, were resolved by electrophoresis through an SDS 7.5-12.5% polyacrylamide gel and transferred to a
filter (Protran BA 85, Schleicher & Schüll). Filters were
incubated with a primary antibody for 2 h, after 1 h blocking
in PBS with 0.05% Tween 20 and 5% dry milk. Primary antibodies used
were as follows: anti E2F-1 (sc-251, Santa Cruz), anti-RB (14001A,
PharMingen), anti-p130 (sc-317, Santa Cruz), anti-AIM-1 (BD
Biosciences), anti-Stathmin (STC, gift of Andre Sobel), anti-E2F-4
(sc-866 Santa Cruz), and anti-B23 (sc-6013, Santa Cruz). Binding of the
primary antibody was detected using an enhanced chemiluminescence
kit (ECL, Amersham Biosciences).
Cell Cycle Flow Cytometry Assays--
Cells were trypsinized and
fixed with methanol ( Chromatin Immunoprecipitation (ChIP)--
Approximately
108 cells were cross-linked by addition of formaldehyde
directly to the growth medium (final concentration 1%). Cross-linking
was stopped after 10 min at room temperature by the addition of glycine
(final concentration: 0.125 M). Cross-linked cells were
washed with PBS, trypsinized, scrapped, washed with PBS, and then
resuspended in buffer I (10 mM HEPES, pH 6.5, 10 mM EDTA, 0.5 mM EGTA, and 0.25% Triton X-100).
Cells were pelleted by microcentrifugation and then resuspended in
buffer II (10 mM HEPES, pH 6.5, 1 mM EDTA, 0.5 mM EGTA, and 200 mM NaCl). After microcentrifugation, nuclei were resuspended in lysis buffer (50 mM Tris, pH 8.1, 10 mM EDTA, 1% SDS, and
protease inhibitors). The resulting chromatin was sonicated to an
average size of 1000 bp and then microcentrifuged. The supernatant was
diluted 1:10 with dilution buffer (10 mM Tris, pH 8.1, 150 mM NaCl, 2 mM EDTA, and 1% Triton X-100) and
divided into aliquots. After preclearing with blocked protein
A-Sepharose beads, 1 µg of antibody was added to each aliquot of
chromatin and incubated on a rotating platform overnight at 4 °C.
Immunocomplexes were recovered with blocked protein A-Sepharose beads.
Following extensive washing, bound DNA fragments were eluted and
analyzed by subsequent PCR. Antibodies used were as follows: anti-E2F-4
(sc-866, Santa Cruz) and anti-p130 (sc-317, Santa Cruz). Primers used
for PCR were for Stathmin promoter (forward) 5'-ACAAGCTGCCGTGTGTCCG-3'
and (reverse) 5'-CTGGAGAGAAGCATTTCGGG-3' and for Our initial studies aimed at understanding the regulation of
mitotic genes by E2F focused on one of these E2F-regulated mitotic genes, Stathmin (also known as oncoprotein 18), which encodes a protein
involved in microtubule dynamics and spindle assembly (44). To
determine whether Stathmin is a direct target of E2F, we infected
NIH3T3 cells with a retrovirus carrying E2F-1 fused to the estrogen
receptor ligand-binding domain (ER-E2F-1). The ER-E2F-1 is expressed as
an inactive fusion protein, which is activated upon addition of the
ligand 4-hydroxytamoxifen (24). As was previously reported by Ishida
et al. (33) induction of E2F-1 led to an increase in
Stathmin mRNA levels (Fig.
1A). Interestingly, a similar
E2F1-induced increase in Stathmin mRNA levels was detected in the
presence of the protein synthesis inhibitor, cycloheximide (CHX) (Fig. 1A). These data indicate that
de novo protein synthesis is not required for E2F1-induced
up-regulation of the Stathmin mRNA, suggesting that Stathmin is a
direct target of E2F.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-E-MLV packaging plasmid and
pBABE-ER-E2F-1 were described previously (24, 42). pBABE-E2F-1dlTA was
generated by inserting the E2F-1dlTA XbaI-HindIII
fragment from pRcCMV-E2F-1-(1-363) (43) into the pBABE-puro vector.
ecotropic packaging plasmid,
pSV-
-E-MLV, and 10 µg of the relevant plasmid using the calcium
phosphate method in the presence of chloroquin (25 µM
final concentration, Sigma C6628). After 8 h, the transfection
medium was replaced with fresh medium, and 5 ml of
retroviral-containing cell supernatant was collected at 6-h intervals.
Five collections were pooled together and frozen in aliquots. For
infection, NIH3T3 cells were incubated for 5 h at 37 °C in 3 ml
of retroviral supernatant, supplemented with 8 µg/ml polybrene
(Sigma H9268). Then, 7 ml of medium was added, and after 24 h the
medium was replaced with fresh medium containing 10% serum and 2 µg/ml puromycin (Sigma P7130).
20 °C). After fixation, cells were
centrifuged for 5 min at 1200 rpm, resuspended in PBS and incubated for
30 min at 4 °C. After recentrifugation cells were resuspended in PBS
containing 5 µg/ml propidium iodide and 50 µg/ml RNase A and
incubated for 30 min at room temperature. Fluorescence intensity was
analyzed using a BD Biosciences flow cytometer.
-actin (forward)
5'-ACTCTTCCAGCCTTCCTTCC-3' and (reverse) 5'-TCCTTCTGCATCCTGTCAGC-3'.
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
View larger version (26K):
[in a new window]
Fig. 1.
Stathmin is a direct E2F target, and its
expression is up-regulated by transcriptionaly inactive E2F-1.
A, NIH3T3 cells were infected with a retrovirus containing
E2F-1 fused to the estrogen receptor ligand-binding domain
(ER-E2F-1). After selection, the cells were kept in medium
containing 0.5% bovine calf serum for 48 h. Then the fusion
protein was activated by the addition of 4-hydroxytamoxifen
(OHT) for the indicated times (in hours), in the absence or
presence of cycloheximide (+CHX). Total RNA was extracted
and used for RT-PCR performed with specific primers for the Stathmin
and GAPDH genes. B, rat fibroblasts containing inducible
wtE2F-1 (E2F-1) or an inducible E2F-1 mutant that lacks the
transactivation and RB-binding domains (E2F-1dlTA)
were kept in medium with 0.1% fetal calf serum for 48 h. Then E2F
expression was induced with 100 µM ZnCl2 for
12 h (+) or not induced ( ) prior to extraction of total RNA.
RT-PCR was performed on the total RNA using specific primers for the
Stathmin and ARPP-PO genes. C, cells were treated as
described in B and then lysed. Protein extracts were
subjected to Western blot analysis using an anti-E2F-1
antibody.
The observed E2F1-induced up-regulation of Stathmin may be due to either activation or derepression. To distinguish between these two possibilities we analyzed the expression of Stathmin in cells containing an inducible wild type E2F-1, or mutant E2F-1, E2F-1dlTA. This truncated E2F-1dlTA lacks both the transactivation and the RB-binding domains, and when overexpressed it can negate both activation of gene expression by E2F and repression by E2F-RB complexes. Once again, induction of wtE2F-1 resulted in an increase in Stathmin mRNA levels (Fig. 1B). Importantly, induction of E2F-1dlTA led to a similar increase in Stathmin mRNA levels (Fig. 1B). wt and mutant E2F-1 were expressed at comparable levels (Fig. 1C). Expression of other E2F-regulated mitotic genes, including Cdc2, BUB1b, EB1, and SAK-a, was similarly up-regulated by both wtE2F-1 (35) and E2F-1dlTA (data not shown). These data strongly suggest that Stathmin, as well as other mitotic genes, is under E2F-dependent negative regulation.
A physiological setting in which such negative regulation may be important is during growth arrest in response to DNA damage.
Low doses of DNA-damaging agents induce cellular growth arrest, and we
analyzed the effects of such a treatment on expression of both Stathmin
and another E2F-regulated mitotic gene, AIM-1. AIM-1(aurora
and Ipl-1-like midbody-associated protein
kinase, also called aurora1 or STK12) is a serine/threonine kinase
required for cytokinesis (reviewed in Refs. 45 and 46). Following
addition of the chemotherapeutic agent doxorubicin, NIH3T3 cells
underwent growth arrest at the G1 and G2/M
phases of the cell cycle (Fig. 2A). This DNA damage-induced
growth arrest was already apparent 24 h after doxorubicin addition
and persisted for at least 72 h. The growth arrest was accompanied
by accumulation of hypophosphorylated RB and p130 (Fig. 2B).
In addition, treatment with doxorubicin led to a significant decrease
in mRNA levels of Stathmin and AIM-1 (Fig. 2C). Similar
results were obtained using HCT116 human colorectal carcinoma cells
(data not shown). The decrease in mRNA levels of the studied genes
could be detected 24 h after treatment and was most evident at
48 h (Fig. 2C). Thus, the increase in
hypophosphorylated RB and p130, which are the growth repressive forms,
coincided with or preceded the down-regulation of Stathmin and AIM-1.
This result raises the possibility that complexes containing either RB
or p130 mediate the repression of Stathmin and AIM-1 in response to DNA
damage.
|
Next we tested whether E2F mediates the decrease in expression of these mitotic genes in response to DNA damage. To this end, NIH3T3 cells were infected with retroviruses containing either E2F-1dlTA or an empty vector and then treated with doxorubicin.
As shown earlier for uninfected cells (Fig. 2), in vector-infected
NIH3T3 cells, the doxorubicin-induced growth arrest was accompanied by
a significant increase in levels of hypophosphorylated RB and p130
(Fig. 3B). In addition, as
shown earlier for uninfected cells, treatment of empty vector-infected
NIH3T3 cells with doxorubicin led to a decrease in mRNA levels of
the E2F-regulated genes, AIM-1 and Stathmin (Fig. 3A). A
similar doxorubicin-induced decrease was detected in protein levels of
AIM-1 and Stathmin (Fig. 3B). Expression of E2F1-dlTA did
not significantly affect the doxorubicin-induced accumulation of
hypophosphorylated RB and p130; however, it abolished the decrease in
mRNA and protein levels of AIM-1 and Stathmin (Fig. 3, A
and B). Thus, in cells expressing E2F1-dlTA, levels of AIM-1
and Stathmin remained unchanged throughout the experiment.
|
Following doxorubicin addition, cells infected with an empty vector, similarly to uninfected cells, arrested at the G1 and G2/M phases of the cell cycle. This arrest at G1 and G2/M persisted for at least 96 h (Fig. 3C). Expression of E2F-1dlTA did not result in noticeable changes in cell cycle distribution of untreated cells; however, it had profound effects on cell cycle distribution after treatment with doxorubicin. Cells expressing E2F-1dlTA failed to arrest at G1 and accumulated at G2/M. Interestingly, growth arrest at G2/M was not maintained, the percentage of cells at G2/M gradually decreased, and a concomitant increase in cells with <2 N and >4 N DNA content was detected (Fig. 3C). These data suggest that while E2F is not essential for the initiation of G2/M arrest, it is required for its maintenance.
To study more directly the role of E2F-RB complexes in the response to
DNA damage, we tested the effect of their dissociation by the papilloma
virus E7 protein. To this end, NIH3T3 cells were infected with a
retrovirus containing either an empty vector, the wtE7 gene of HPV16,
or a mutated E7, E721-35, which does not bind RB family members.
The wt and mutated E7 were expressed at similar levels (data not
shown); however, expression of wtE7, but not E7
21-35, inhibited the
doxorubicin-induced decrease in protein levels of AIM-1 and Stathmin
(Fig. 4A). In addition,
expression of wtE7 led to profound changes in cell cycle distribution
after doxorubicin treatment (Fig. 4B). The effects of wtE7
expression were highly similar to those of E2F-1dlTA and included: 1)
failure of cells to arrest at G1 and their accumulation at
G2/M; 2) inability to maintain growth arrest at
G2/M, with a gradual decrease in the percentage of cells at
G2/M and an increase in cells with <2 N and
>4 N DNA content (Fig. 4B). Expression of
E7
21-35 had no apparent effect on cell cycle distribution after DNA
damage (Fig. 4B). These findings suggest that endogenous
E2F-RB complexes mediate repression of mitotic genes and sustained
G2/M arrest in response to DNA damage.
|
To identify the distinct members of the E2F and RB families
that mediate the repression of E2F-regulated genes in response to DNA
damage we performed a ChIP using antibodies directed against specific
family members. The human Stathmin promoter contains three putative
E2F-binding sites at positions 28,
577, and
701 upstream to the
transcription start site (47), and our analysis of the murine Stathmin
promoter indicates that it too contains three putative E2F-binding
sites. Taken together with our observation that E2F-induced
up-regulation of Stathmin does not require de novo protein
synthesis (Fig. 1A), this sequence information suggests that
E2Fs may interact with the Stathmin promoter. Therefore, we analyzed
this promoter for occupancy by E2F and RB family members. We observed a
significant enrichment of the Stathmin promoter when using E2F-4 or
p130 antibodies (Fig. 5A). We
did not detect any enrichment after amplification of an unrelated
genomic DNA fragment (Fig. 5A). The enrichment of the
Stathmin promoter when using p130 and E2F-4 antibodies was
more prominent in cells treated with doxorubicin (Fig.
5B), indicating an increase in the occupancy of this
promoter by E2F-4 and p130 after genotoxic stress. In agreement with
this observation, we detected an increase in levels of E2F-4 and
hypophosphorylated p130 in the nuclei of cells treated with doxorubicin
(Fig. 5C). Taken together, our data indicate that endogenous
E2F-RB complexes down-regulate expression of the mitotic genes AIM-1
and Stathmin in response to DNA damage. Furthermore, our findings
strongly suggest that E2F-RB complexes play a role in the maintenance
of DNA damage-induced G2/M arrest.
|
![]() |
DISCUSSION |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Recent screens aimed at identifying novel genes regulated by E2F suggest that E2F modulates the expression of a number of genes involved in entry to and progression through mitosis as well as genes that affect the G2/M checkpoint (33, 35, 36). We show here that two pivotal mitotic regulators, Stathmin and AIM-1, are subjected to E2F-mediated negative regulation, and their expression is elevated by a dominant negative mutant of E2F-1 that lacks the transactivation and RB-binding domains.
Stathmin/oncoprotein 18 is a conserved cytoplasmic phosphoprotein that physically interacts with tubulin dimers (reviewed in Ref. 44). It is a critical regulator of microtubule dynamics during cell cycle progression and governs preferential microtubule growth around chromosomes during spindle assembly (48). Stathmin is expressed at elevated levels in various human tumors (49, 50), and it is expressed at higher levels in proliferating cells as compared with non-proliferating cells (51). The Stathmin promoter contains three E2F-binding sites (47), and a recent study indicates that Stathmin levels are regulated by E2F (33). Importantly, antisense inhibition of Stathmin expression results in growth arrest and accumulation of cells in G2/M (52, 53). Conversely, overexpression of Stathmin abrogates irradiation-induced G2/M arrest. Thus, Stathmin appears to have an essential role in the G2/M arrest mechanism (54).
The second E2F-regulated gene studied here, AIM-1, is an
Aurora/Ipl1p-related serine/threonine kinase that is required for cytokinesis (reviewed in Refs. 45 and 46). Regulation of AIM-1 expression is not well characterized, and its promoter has not been
studied extensively. Analysis of genomic sequences upstream to human
AIM-1 coding sequence reveals an E2F-binding site at 61 upstream to
the putative transcription start site, which is conserved in mouse
AIM-1. Furthermore, E2F-4 was shown to interact with the human
AIM-1 promoter (36).
AIM-1 expression is cell cycle-regulated, and its mRNA and protein accumulate at G2/M (55-57). The kinase activity of AIM-1 is also cell cycle-regulated with peak activity at the M phase (58). The intracellular localization of AIM-1 is consistent with a role in the later stages of mitosis as it is found in the central spindles of anaphase cells and at the midbody of telophase cells (56, 58). Both expression of a kinase-inactive form of AIM-1 and overexpression of wt AIM-1 block cytokinesis and lead to polyploidity, indicating that proper AIM-1 expression and activity is critical for cytokinesis (55, 56).
The results presented here demonstrate that expression of AIM-1 and Stathmin is repressed in response to DNA damage. We show that this repression is abrogated by dominant negative E2F-1 as well as by dissociation of E2F-RB complexes, indicating that such complexes play a critical role in the regulation of these genes. In support of this notion, we detected in vivo binding of E2F-4 and p130 to the Stathmin promoter. Importantly, DNA damage leads to an increase in levels of nuclear E2F-4 and p130 and enhancement of their binding to the Stathmin promoter. Of note, overexpression of p53 was shown to increase in vitro binding of E2F-4 and p130 to the promoter of cdc2, another E2F-regulated mitotic gene (59).
Our data also implicate E2F in maintenance of induced G2/M arrest and indicate that functional E2F-RB complexes are required to sustain G2/M arrest following genotoxic stress. Treatment of unmanipulated cells and cells expressing mutated E7 (that does not dissociate E2F-RB complexes) with doxorubicin resulted in a sustained arrest at G1 and G2/M. In sharp contrast, doxorubicin treatment of cells expressing either E7 (which dissociates E2F-RB complexes) or E2F-1dlTA (which competes with E2F-RB complexes for DNA binding) led to a transient G2/M arrest. Furthermore, in cells expressing E7 or E2F-1dlTA, the premature exit from G2 after genotoxic stress was accompanied by appearance of cells with >4 N DNA content, indicating that loss of functional E2F and E2F-RB complexes may result in endoreduplication. This is in agreement with the suggested role of AIM-1 in maintaining cell diploidity and with previous studies demonstrating that lack of RB is correlated with increased endoreduplication (60). The data presented here further support a role for endogenous E2F in preventing endoreduplication after DNA damage.
Overall, the data presented here demonstrate that E2F-RB complexes play
an important role both in maintaining G2/M arrest and in
repression of two mitotic genes after DNA damage. The complete panel of
genes that are repressed by E2F-RB complexes and their relative
contribution to sustained G2/M arrest remain to be
determined. Cdc2 and cyclin B1 are two E2F-regulated genes (32,
33) whose promoter activity was shown to be down-regulated upon
sustained G2/M arrest or p53 activation (59, 61, 62).
Protein levels of cdc2 and cyclin B1 as well as cyclin B1/cdc2 kinase
activity also decrease in association with sustained G2/M
arrest after DNA damage (14, 59, 61), and this decrease is abrogated by
E7, implicating the RB family in their negative regulation. These data
strongly suggest that upon DNA damage, expression of both cyclin B1 and
cdc2 is repressed by E2F-RB complexes. However, there is conflicting
data regarding the dependence of sustained G2/M arrest on
reduction of cyclin B1/cdc2 activity. While one study demonstrated that
constitutive activation of cyclin B1/cdc2 kinase activity overrides
p53-mediated G2/M arrest (63), others detected prolonged
G2/M arrest after DNA damage even in cells with high cyclin
B1/cdc2 activity (61). Thus, additional genes repressed by E2F-RB
complexes probably play a pivotal role in sustaining G2/M
arrest. A number of additional E2F-regulated genes, including cyclin A,
thymidine kinase, topoisomerase II, and RAD51 are repressed upon DNA
damage (64). We show here that the E2F-dependent decrease
in expression of two genes involved in mitosis, AIM-1 and Stathmin, is
associated with sustained G2/M arrest after DNA damage. It
is tempting to speculate that sustaining G2 arrest after
DNA damage involves the concerted, E2F-mediated, repression of a large
panel of genes.
![]() |
ACKNOWLEDGEMENTS |
---|
We thank Yocheved Lamed for excellent
technical assistance. We thank Moshe Oren for pBABE-E7 and
pBABE-E721-35, Kristian Helin for pBABE-ER-E2F-1 and Andre Sobel
for the STC anti Stathmin antibody. We thank Nick Dyson and Kristian
Helin for critical reading of the manuscript.
![]() |
FOOTNOTES |
---|
* This work was supported by the Israel Cancer Research Fund (ICRF) and Yad Abraham Research Center for Diagnostics and Therapy.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.
Incumbent of the Recanati Career Development chair of cancer
research. To whom correspondence should be addressed: Dept. of Molecular Cell Biology, The Weizmann Inst. of Science, Rehovot 76100, Israel. Tel.: 972-8-934-2239; Fax: 972-8-934-4125; E-mail: doron. ginsberg{at}weizmann.ac.il.
Published, JBC Papers in Press, November 21, 2002, DOI 10.1074/jbc.M210327200
![]() |
ABBREVIATIONS |
---|
The abbreviations used are: RB, retinoblastoma; ER, estrogen receptor; RT, reverse transcription; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; PBS, phosphate-buffered saline; ChIP, chromatin immunoprecipitation; wt, wild type; FACS, fluorescence-activated cell sorter.
![]() |
REFERENCES |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
1. | Zhou, B. B., and Elledge, S. J. (2000) Nature 408, 433-439[CrossRef][Medline] [Order article via Infotrieve] |
2. | Khanna, K. K., and Jackson, S. P. (2001) Nat. Genet. 27, 247-254[CrossRef][Medline] [Order article via Infotrieve] |
3. | Agami, R., and Bernards, R. (2000) Cell 102, 55-66[Medline] [Order article via Infotrieve] |
4. | Costanzo, V., Robertson, K., Ying, C. Y., Kim, E., Avvedimento, E., Gottesman, M., Grieco, D., and Gautier, J. (2000) Mol. Cell 6, 649-659[Medline] [Order article via Infotrieve] |
5. |
Mailand, N.,
Falck, J.,
Lukas, C.,
Syljuasen, R. G.,
Welcker, M.,
Bartek, J.,
and Lukas, J.
(2000)
Science
288,
1425-1429 |
6. |
Peng, C. Y.,
Graves, P. R.,
Thoma, R. S., Wu, Z.,
Shaw, A. S.,
and Piwnica-Worms, H.
(1997)
Science
277,
1501-1505 |
7. |
Sanchez, Y.,
Wong, C.,
Thoma, R. S.,
Richman, R., Wu, Z.,
Piwnica-Worms, H.,
and Elledge, S. J.
(1997)
Science
277,
1497-1501 |
8. |
Brugarolas, J.,
Moberg, K.,
Boyd, S. D.,
Taya, Y.,
Jacks, T.,
and Lees, J. A.
(1999)
Proc. Natl. Acad. Sci. U. S. A.
96,
1002-1007 |
9. | Deng, C., Zhang, P., Harper, J. W., Elledge, S. J., and Leder, P. (1995) Cell 82, 675-684[Medline] [Order article via Infotrieve] |
10. | Waldman, T., Kinzler, K. W., and Vogelstein, B. (1995) Cancer Res. 55, 5187-5190[Abstract] |
11. |
Bunz, F.,
Dutriaux, A.,
Lengauer, C.,
Waldman, T.,
Zhou, S.,
Brown, J. P.,
Sedivy, J. M.,
Kinzler, K. W.,
and Vogelstein, B.
(1998)
Science
282,
1497-1501 |
12. | Harper, J. W., Adami, G. R., Wei, N., Keyomarsi, K., and Elledge, S. J. (1993) Cell 75, 805-816[Medline] [Order article via Infotrieve] |
13. |
Harrington, E. A.,
Bruce, J. L.,
Harlow, E.,
and Dyson, N.
(1998)
Proc. Natl. Acad. Sci. U. S. A.
95,
11945-11950 |
14. |
Flatt, P. M.,
Tang, L. J.,
Scatena, C. D.,
Szak, S. T.,
and Pietenpol, J. A.
(2000)
Mol. Cell. Biol.
20,
4210-4223 |
15. |
Dyson, N.
(1998)
Genes Dev.
12,
2245-2262 |
16. | Nevins, J. R. (1998) Cell Growth Differ. 9, 585-593[Medline] [Order article via Infotrieve] |
17. |
Harbour, J. W.,
and Dean, D. C.
(2000)
Genes Dev.
14,
2393-2409 |
18. | Zhang, H. S., and Dean, D. C. (2001) Oncogene 20, 3134-3138[CrossRef][Medline] [Order article via Infotrieve] |
19. | Moberg, K., Starz, M. A., and Lees, J. A. (1996) Mol. Cell. Biol. 16, 1436-1449[Abstract] |
20. | Johnson, D. G., Schwarz, J. K., Cress, W. D., and Nevins, J. R. (1993) Nature 365, 349-352[CrossRef][Medline] [Order article via Infotrieve] |
21. |
Qin, X. Q.,
Livingston, D. M.,
Kaelin, W. G., Jr.,
and Adams, P. D.
(1994)
Proc. Natl. Acad. Sci. U. S. A.
91,
10918-10922 |
22. | Lukas, J., Petersen, B. O., Holm, K., Bartek, J., and Helin, K. (1996) Mol. Cell. Biol. 16, 1047-1057[Abstract] |
23. |
DeGregori, J.,
Leone, G.,
Miron, A.,
Jakoi, L.,
and Nevins, J. R.
(1997)
Proc. Natl. Acad. Sci. U. S. A.
94,
7245-7250 |
24. |
Vigo, E.,
Muller, H.,
Prosperini, E.,
Hateboer, G.,
Cartwright, P.,
Moroni, M. C.,
and Helin, K.
(1999)
Mol. Cell. Biol.
19,
6379-6395 |
25. | Kowalik, T. F., DeGregori, J., Leone, G., Jakoi, L., and Nevins, J. R. (1998) Cell Growth Differ. 9, 113-118[Abstract] |
26. |
Takahashi, Y.,
Rayman, J. B.,
and Dynlacht, B. D.
(2000)
Genes Dev.
14,
804-816 |
27. | Trimarchi, J. M., and Lees, J. A. (2002) Nat. Rev. Mol. Cell. Biol. 3, 11-20[CrossRef][Medline] [Order article via Infotrieve] |
28. |
Magae, J., Wu, C. L.,
Illenye, S.,
Harlow, E.,
and Heintz, N. H.
(1996)
J. Cell Sci.
109,
1717-1726 |
29. |
Lindeman, G. J.,
Gaubatz, S.,
Livingston, D. M.,
and Ginsberg, D.
(1997)
Proc. Natl. Acad. Sci. U. S. A.
94,
5095-5100 |
30. | Muller, H., Moroni, M. C., Vigo, E., Petersen, B. O., Bartek, J., and Helin, K. (1997) Mol. Cell. Biol. 17, 5508-5520[Abstract] |
31. | Verona, R., Moberg, K., Estes, S., Starz, M., Vernon, J. P., and Lees, J. A. (1997) Mol. Cell. Biol. 17, 7268-7282[Abstract] |
32. | Tommasi, S., and Pfeifer, G. P. (1995) Mol. Cell. Biol. 15, 6901-6913[Abstract] |
33. |
Ishida, S.,
Huang, E.,
Zuzan, H.,
Spang, R.,
Leone, G.,
West, M.,
and Nevins, J. R.
(2001)
Mol. Cell. Biol.
21,
4684-4699 |
34. |
Muller, H.,
Bracken, A. P.,
Vernell, R.,
Moroni, M. C.,
Christians, F.,
Grassilli, E.,
Prosperini, E.,
Vigo, E.,
Oliner, J. D.,
and Helin, K.
(2001)
Genes Dev.
15,
267-285 |
35. | Polager, S., Kalma, Y., Berkovich, E., and Ginsberg, D. (2002) Oncogene 21, 437-446[CrossRef][Medline] [Order article via Infotrieve] |
36. |
Ren, B.,
Cam, H.,
Takahashi, Y.,
Volkert, T.,
Terragni, J.,
Young, R. A.,
and Dynlacht, B. D.
(2002)
Genes Dev.
16,
245-256 |
37. | Huang, Y., Ishiko, T., Nakada, S., Utsugisawa, T., Kato, T., and Yuan, Z. M. (1997) Cancer Res. 57, 3640-3643[Abstract] |
38. |
Blattner, C.,
Sparks, A.,
and Lane, D.
(1999)
Mol. Cell. Biol.
19,
3704-3713 |
39. |
Hofferer, M.,
Wirbelauer, C.,
Humar, B.,
and Krek, W.
(1999)
Nucleic Acids Res.
27,
491-495 |
40. | O'Connor, D. J., and Lu, X. (1999) Oncogene 19, 2369-2376[CrossRef] |
41. |
Lin, W. C.,
Lin, F. T.,
and Nevins, J. R.
(2001)
Genes Dev.
15,
1833-1844 |
42. | Muller, A. J., Young, J. C., Pendergast, A. M., Pondel, M., Landau, N. R., Littman, D. R., and Witte, O. N. (1991) Mol. Cell. Biol. 11, 1785-1792[Medline] [Order article via Infotrieve] |
43. | Hofmann, F., Martelli, F., Livingston, D. M., and Wang, Z. (1996) Genes Dev. 10, 2949-2959[Abstract] |
44. | Cassimeris, L. (2002) Curr. Opin. Cell Biol. 14, 18-24[CrossRef][Medline] [Order article via Infotrieve] |
45. | Bischoff, J. R., and Plowman, G. D. (1999) Trends Cell Biol. 9, 454-459[CrossRef][Medline] [Order article via Infotrieve] |
46. | Terada, Y. (2001) Cell Struct. Funct. 26, 653-657[CrossRef][Medline] [Order article via Infotrieve] |
47. |
Melhem, R. F.,
Zhu, X. X.,
Hailat, N.,
Strahler, J. R.,
and Hanash, S. M.
(1991)
J. Biol. Chem.
266,
17747-17753 |
48. | Andersen, S. S., Ashford, A. J., Tournebize, R., Gavet, O., Sobel, A., Hyman, A. A., and Karsenti, E. (1997) Nature 389, 640-643[CrossRef][Medline] [Order article via Infotrieve] |
49. |
Hanash, S. M.,
Strahler, J. R.,
Kuick, R.,
Chu, E. H.,
and Nichols, D.
(1988)
J. Biol. Chem.
263,
12813-12815 |
50. | Bieche, I., Lachkar, S., Becette, V., Cifuentes-Diaz, C., Sobel, A., Lidereau, R., and Curmi, P. A. (1998) Br. J. Cancer 78, 701-709[Medline] [Order article via Infotrieve] |
51. | Rowlands, D. C., Williams, A., Jones, N. A., Guest, S. S., Reynolds, G. M., Barber, P. C., and Brown, G. (1995) Lab. Invest. 72, 100-113[Medline] [Order article via Infotrieve] |
52. |
Luo, X. N.,
Mookerjee, B.,
Ferrari, A.,
Mistry, S.,
and Atweh, G. F.
(1994)
J. Biol. Chem.
269,
10312-10318 |
53. |
Marklund, U.,
Osterman, O.,
Melander, H.,
Bergh, A.,
and Gullberg, M.
(1994)
J. Biol. Chem.
269,
30626-30635 |
54. | Johnsen, J. I., Aurelio, O. N., Kwaja, Z., Jorgensen, G. E., Pellegata, N. S., Plattner, R., Stanbridge, E. J., and Cajot, J. F. (2000) Int. J. Cancer 88, 685-691[CrossRef][Medline] [Order article via Infotrieve] |
55. | Tatsuka, M., Katayama, H., Ota, T., Tanaka, T., Odashima, S., Suzuki, F., and Terada, Y. (1998) Cancer Res. 58, 4811-4816[Abstract] |
56. |
Terada, Y.,
Tatsuka, M.,
Suzuki, F.,
Yasuda, Y.,
Fujita, S.,
and Otsu, M.
(1998)
EMBO J.
17,
667-676 |
57. |
Kawasaki, A.,
Matsumura, I.,
Miyagawa, J.,
Ezoe, S.,
Tanaka, H.,
Terada, Y.,
Tatsuka, M.,
Machii, T.,
Miyazaki, H.,
Furukawa, Y.,
and Kanakura, Y.
(2001)
J. Cell Biol.
152,
275-287 |
58. |
Bischoff, J. R.,
Anderson, L.,
Zhu, Y.,
Mossie, K., Ng, L.,
Souza, B.,
Schryver, B.,
Flanagan, P.,
Clairvoyant, F.,
Ginther, C.,
Chan, C. S.,
Novotny, M.,
Slamon, D. J.,
and Plowman, G. D.
(1998)
EMBO J.
17,
3052-3065 |
59. |
Taylor, W. R.,
Schonthal, A. H.,
Galante, J.,
and Stark, G. R.
(2001)
J. Biol. Chem.
276,
1998-2006 |
60. |
Niculescu, A. B., III,
Chen, X.,
Smeets, M.,
Hengst, L.,
Prives, C.,
and Reed, S. I.
(1998)
Mol. Cell. Biol.
18,
629-643 |
61. |
Passalaris, T. M.,
Benanti, J. A.,
Gewin, L.,
Kiyono, T.,
and Galloway, D. A.
(1999)
Mol. Cell. Biol.
19,
5872-5881 |
62. |
Manni, I.,
Mazzaro, G.,
Gurtner, A.,
Mantovani, R.,
Haugwitz, U.,
Krause, K.,
Engeland, K.,
Sacchi, A.,
Soddu, S.,
and Piaggio, G.
(2001)
J. Biol. Chem.
276,
5570-5576 |
63. |
Park, M.,
Chae, H. D.,
Yun, J.,
Jung, M.,
Kim, Y. S.,
Kim, S. H.,
Han, M. H.,
and Shin, D. Y.
(2000)
Cancer Res.
60,
542-545 |
64. | de Toledo, S. M., Azzam, E. I., Keng, P., Laffrenier, S., and Little, J. B. (1998) Cell Growth Differ. 9, 887-896[Abstract] |