(Received for publication, August 4, 1995; and in revised form, September 22, 1995)
From the
Exposure of mammalian cells to ionizing radiation results in the
induction of the immediate early genes, c-jun and Egr-1, which encode transcription factors implicated in cell
growth as well as the cellular response to oxidative stress. We studied
the role of these immediate early genes in cell cycle kinetics and cell
survival following x-irradiation of clones containing inducible
dominant negatives to c-jun and Egr-1. The dominant
negative constructs to c-jun (9) and Egr-1 (WT/Egr) prevented x-ray induction of transcription through the
AP-1 and Egr binding sites, respectively. Twenty percent of confluent,
serum-deprived SQ20B human tumor cells, normal fibroblasts, and
fibroblasts from patients with ataxia telangiectasia entered S phase
within 5 h of irradiation. Clones containing inducible
9 and
WT/Egr dominant negative constructs demonstrated attenuation of the
percentage of cells exiting G
phase and reduced survival
following irradiation. These data indicate that the dominant negatives
to the stress-inducible immediate early genes Egr-1 and
c-jun prevent the onset of S phase and reduce the survival of
human cells exposed to ionizing radiation.
Although DNA damage-induced cell cycle delay has been the focus
of recent
reports(1, 2, 3, 4, 5) ,
certain aspects of the cellular response to ionizing radiation resemble
a growth-like response. For example, the kinetics of tumor and normal
tissue cell repopulation is accelerated after
irradiation(6, 7, 8) . Cell survival and
proliferation following x-irradiation are in part regulated by growth
factors and cytokines that are induced by ionizing
radiation(9, 10, 11, 12) .
Furthermore, a recent report demonstrates that when irradiated, cells
beyond the G restriction point enter S phase(3) .
To study the proliferative response to x-irradiation, we irradiated
serum-deprived cells and determined whether x-irradiation initiates DNA
synthesis in a subpopulation of G
phase cells. Following a
transient G
delay, a reduction in the percentage of cells
in G
was evident within 2 h after irradiation. This
reduction was temporally related to c-jun and Egr-1 expression.
Egr-1 and c-jun are immediate
early genes encoding transcription factors that are implicated in the
response of cells to a variety of stressful
stimuli(13, 14, 15, 16, 17) .
Induction of these genes is associated with cell growth as well as the
cellular response to oxidative stress (12, 18, 19; reviewed in (20) and (21) ). The transcriptional regulation of
genes in response to exposure to ionizing radiation results in the
expression of many genes, including Egr-1 and
c-jun(14, 15, 22) . The role of
these genes in the cellular response to x-rays may be the recruitment
of quiescent cells into the cell cycle and subsequent repopulation of
tissues exposed to oxidative stress(23) . In particular,
c-jun is a protooncogene encoding the transcription factor
Jun, a component of the AP-1 protein complex which regulates the
transcription of a number of genes, including growth factors and
cytokines (reviewed in (13) ). We therefore studied the role of Egr-1 and c-jun in the radiation response by using of
dominant negative constructs to Egr-1 and c-jun. The
c-jun dominant negative (9) was created by replacing the
transcriptional activation domain of c-jun with the
transcriptional inhibitory domain of Lex A, while maintaining the DNA
binding region(24) . The mutant Jun lacks an
activation domain and blocks stimulation of transcription by several
oncoproteins, including Ras and v-Src, as well as by phorbol esthers.
We constructed the Egr-1 dominant negative by replacing the
transcriptional regulatory domain with that of a negative
transcriptional regulator of WT-1(25) . The repression
function was mapped to the glutamine- and proline-rich NH
terminus of WT1; fusion of this domain to the zinc
finger region of Egr-1 converted Egr-1 into a
transcriptional repressor. This construct (WT/Egr) binds to
the Egr-1 binding site (EBS) (
)to block the
transcriptional activation of downstream genes regulated by EBS
cis-acting elements. We found that WT/Egr and
9 prevented
x-ray induction of EBS-CAT and AP1-CAT reporter constructs and abated
G
/S transition after x-irradiation.
Ataxia
telangiectasia (AT) is an autosomal recessive disorder characterized by
progressive ataxia, immunodeficiency, a predisposition to the
development of cancer, and marked radiosensitivity (reviewed in (26) ). Cells isolated from patients with some AT complementary
groups demonstrate neither inhibition of DNA synthesis nor G delay following exposure to ionizing radiation (27) . We
found that radiation-mediated c-jun and Egr-1 expression was prolonged in AT fibroblasts. This unopposed
immediate early gene expression was associated with a prolonged exit
from G
into S phase after irradiation. These data indicate
that persistent DNA damage in x-irradiated AT fibroblasts may provide a
signal for unabated immediate early gene induction and subsequent exit
from G
. We found that the c-jun and Egr-1 dominant negatives also prevented irradiated AT fibroblasts from
exiting G
. These data demonstrate phenotypes for the
x-ray-inducible immediate early genes c-jun and Egr-1.
For creation of stably integrated
transfectants, inducible dominant negative constructs within plasmids
containing the neomycin resistance gene (CMV-Neo) were
transfected into SQ20B, IMR90, and AT5BI cells with Lipofectin reagent
(Life Technologies, Inc.). Stably integrated transfectants were
selected by growth in G418 (800 µg/ml) and trypsinization of
individual colonies by use of cloning cylinders. IMR90 colonies stopped
growing after several divisions and could not be cloned. Similarly
SQ20B colonies containing 9 discontinued growth and could not be
cloned. SQ20B and AT5BI clones containing dominant negatives were
maintained in G418 (800 µg/ml). We determined the inhibition of
x-ray induction of the EBS-CAT or 3
TRE-CAT constructs when
ZnS0
was added. The clone demonstrating the greatest
inhibition was then maintained in G418 and used for FACS and survival
analysis.
Figure 1:
DNA
quantification following irradiation. Confluent, serum-deprived celss
were irradiated and DNA was stained with propidium iodide. The DNA
quantity of cells was quantified by FACS analysis, and the percentages
of cells in G and S were calculated by means of the Cell
Fit program (Becton Dickinson). RNA was extracted at the indicated
times following irradiation. Northern blots were hybridized to
P-labeled c-jun and Egr-1 DNA probes
followed by autoradiography and quantification by densitometry. Data
presented show the mean and S.E. of three to four Northern blots. A, c-jun and Egr-1 gene expression and
percentage of irradiated SQ-20B cells and G
in S phase. B, c-jun and Egr-1 gene expression and the
percentage of IMR-90 fibroblasts in G
in S phase. C, c-jun and Egr-1 gene expression and AT5BI
fibroblasts in G
and S phase.
We next examined AT
fibroblasts that are known to be deficient in G delay. As
with non-AT cells, 15% of AT fibroblasts (AT5BI) exited G
and accumulated in S phase within 5 h, but exit from G
continued for 9 h, with a total of a 22% reduction in the
percentage of cells in G
(Fig. 1C). These
findings are supported by the recent report that cells beyond the
G
restriction point enter S phase following
x-irradiation(3) .
Immediate
early gene expression in irradiated AT5BI cells was also analyzed.
Expression of the c-jun gene increased 8-fold at 30 min,
whereas Egr-1 expression increased 9-fold. Increased
expression of both Egr-1 and c-jun persisted for 9 h
following irradiation of the AT cells, in contrast to normal
fibroblasts and tumor cell lines in which immediate early gene
induction was transient (Fig. 1). The prolonged immediate early
gene expression following irradiation was associated with prolongation
of the G/S transition. These data indicate that ionizing
radiation-induced exit from G
and entry into S phase are
temporally associated with immediate early gene induction in a manner
analogous to their expression following stimulation with mitogens.
Figure 2:
Dominant negative mutants to c-jun and Egr-1 prevent x-ray induction through AP-1 and Egr
binding sites. Cells were cotransfected with the Egr-1 dominant negative (WT/Egr) and the Egr-1 binding site
(EBS) linked to a CAT reporter. Transfectants were irradiated 18 h
later. Shown are CAT enzyme levels calculated as the percent conversion
of [C]chloramphenicol to the acetylated forms.
ATIVB fibroblasts were cotransfected with the c-jun dominant
negative (
9), and the AP-1 binding element linked to a CAT
reporter (3
TRE-CAT). Transfectants were irradiated 18 h later.
Shown are the means and S.E. from three experiments. A, SQ20B
cells transfected wth WT/Egr dominant negative. B, AT5BI
transfected with WT/Egr dominant negative. C, AT5BI
fibroblasts transfected with
9.
To study the role of c-jun induction in the onset of S phase following irradiation, we
utilized the transacting dominant negative to c-jun (9)
in which the DNA binding region is maintained, and the transcriptional
activation domain is replaced with the transcriptional inhibitory
domain of Lex A(24) . We first cotransfected the
9
expression vector with the AP-1-CAT reporter construct (3
TRE) to
determine whether
9 prevented radiation-mediated AP-1 activation.
Both plasmids (pMT-D9 and p3
TRE-CAT) were added to Lipofectin
solution and incubated. A reduction in the radiation inducibility of
AP-1 by
9 was observed (Fig. 2C). A 3-fold
increase in CAT expression resulted from irradiation of AT5BI cells
transfected with 3
TRE-CAT. This increased expression was
attenuated when
9 was cotransfected with 3
TRE-CAT.
Figure 3:
Dominant negative mutants to c-jun and Egr-1 prevent x-ray-mediated exit from G.
Clones containing dominant negatives were grown to confluence for 4
days and serum-deprived for 16 h. They were treated with 5 Gy (GE
Maxitron generator at 2 Gy/min). Clones were then trypsinized and fixed
in cold ethanol followed by staining with propidium iodide. DNA was
quantified with a FACSCANNER (Becton Dickinson), and the percentages of
cells in G
, S, and G
phases were calculated by
means of the Cell Fit program (Becton Dickinson). During induction of
the dominant negatives, ZnSO
(100 µM) was
added for 5 h prior to irradiation. Experiments were performed three to
four times, and the mean and standard error of the mean are shown. A, cell cycle kinetics of irradiated SQ20B clone containing
WT/Egr dominant negative construct. B, cell cycle kinetics of
irradiated AT5BI clone containing WT/Egr dominant negative construct. C, cell cycle kinetics of irradiated AT5BI clone containing
the
9 dominant negative construct
Stably transfected clones containing the
9 dominant negative under the transcriptional control of the
metallothionein promoter were selected for expression of the neomycin
resistance gene. Colonies grown in the presence of G418 demonstrated no
3
TRE induction by x-irradiation. AT clones containing
9
demonstrated attenuated exit from G
when pretreated with
ZnSO
for 4 h prior to irradiation. Clones treated in the
absence of ZnSO
demonstrated a 15% reduction in the
percentage of cells in G
within 9 h after irradiation (Fig. 3C). Exit from G
after x-irradiation
of nontransfected cells was not affected by ZnSO
. A
concomitant increase in the percentage of cells in S phase occurred
during this time interval.
Figure 4:
Dominant negative mutants to c-jun and Egr-1 reduce survival after x-irradiation. Clones
were irradiated with the indicated doses. Plates were stained, and
colonies were counted 14 days following irradiation. ZnSO (100 µM) was added for 5 h prior to irradiation.
Experiments were performed three to four times, and the mean and
standard error of the mean are shown.
To study the effects of c-jun dominant
negative constructs on cell killing by radiation, we irradiated the
clone containing 9 with or without ZnSO
added prior to
the irradiation. The plating efficiency for the AT-
9 clone was 25%
and did not differ from that for wild-type AT5BI fibroblasts or clones
treated with ZnSO
. Radiation survival analysis was
performed with the colony-forming assay. Fig. 4shows that,
following treatment with 5 Gy, the surviving fraction of wild-type
AT5BI cells was 0.0006 and was 0.0002 in
9-containing clones
treated with ZnSO
(p < 0.05). Thus, attenuation
of transcriptional activation through AP-1 is associated with reduced
cell survival following x-irradiation.
Our objective in this study was to determine the role of the
immediate early genes c-jun and Egr-1 in the response
of human cells to ionizing radiation. We found that radiation-mediated
expression of c-jun and Egr-1 preceded the departure
of cells from G. To study the requirement for these genes
during the exit from late G
into S phase, we utilized
inducible dominant negatives to c-jun and Egr-1.
Basal expression of these constructs was observed as diminished
transcriptional activation in the absence of ZnSO
. Each of
the clones that grew in the presence of G418 was tested for inhibition
of x-ray induction of the promoter-reporter constructs (EBS-CAT and
3
TRE-CAT) in the presence of zinc sulfate. These results
indicated the extent to which the dominant negatives are inducible in
these clones. Each clone that demonstrated optimal induction of
dominant negative activity was maintained in G418. FASCS analysis and
radiation survival analysis were performed only on these clones,
demonstrating optimal dominant negative induction by ZnSO
.
We found that dominant negatives to both Egr-1 and c-jun attenuated the G
exit of irradiation cells. These
cells were serum-deprived and confluent, and therefore a large
percentage of them were already in G
. Following a transient
G
delay, irradiated mammalian cells typically demonstrate
an accumulation in S phase (45) (reviewed in (46) ).
This has been referred to as an S phase arrest which is followed by a
more pronounced G
delay. It is important to note that the
percentage of cells in G
did not vary throughout the 8 h of
observation in these experiments. Taken together, these data indicate
that transcriptional regulation through AP-1 and the Egr binding site
in part regulates exit from G
following irradiation. These
findings do not exclude the possibility that these dominant negatives
act through mechanisms other than blocking transcriptional activation.
Jun and Egr-1 may have metabolic functions that have not been
determined. One possible example of this is the demonstration that
c-jun participates in DNA
replication(37, 38) . Furthermore, the transcriptional
activation domain of Jun is required for both transcriptional
activation and DNA synthesis(47) . Regardless of its mechanism
of action, data presented herein indicate that Jun is induced by
ionizing radiation and is required for transition from G
to
S phase.
We found a 1-h delay in G before cells enter S
phase as shown by previous studies(3, 46) . These data
are supported by the recent finding that cells beyond the G
restriction point enter S phase after x-irradiation(3) . Cell
cycle-regulated genes such as thymidine kinase, dihydrofolate
reductase, DNA polymerase-
, and PCNA are all induced during late
G
to early S phase(48) . In addition, the
retinoblastoma susceptibility gene (Rb), as well as genes encoding the
transcription factors E2F1, c-Myb, and Jun, have been shown to be
induced in late G
. Their respective gene products are
presumably involved in the regulation of subsequently expressed genes
required for DNA synthesis. We have found that 20% of cells begin to
synthesize DNA following a brief delay of 1-2 h after
irradiation. The transient G
delay observed in this study
is consistent with that found in previous studies(49) . This
brief delay that precedes the onset of DNA synthesis may be due to the
prerequisite formation of protein complexes such as E2F-pRb or E2F
combined with other cell cycle-regulating elements such as cyclin-E,
cdk2, p107, and p130(50) .
We found that the rapid induction
of c-jun and Egr-1, and the subsequent onset of DNA
replication, were associated with the survival response of mammalian
cells to ionizing radiation. Although there is a correlation between
exit from G and cell survival in the present study, a
causal relationship has not been established. One of the difficulties
in further establishing the impact of dominant negative constructs to
c-jun and Egr-1 on radiation survival is that these
constructs induce cell killing. We found that the c-jun dominant
negative construct (
9) prevented colony formation in both IMR-90
fibroblasts and SQ-20B cells whereas, WT/Egr prevented colony formation
in IMR-90 fibroblasts only. It is not known why SQ-20B cells will grow
following transfection with WT/Egr but do not grow when
9 is
expressed at low basal levels. Although these dominant negative
constructs are regulated by the metallothionein promoter, we found that
there is low basal expression. Even in the absence of ZnSO
,
these constructs reduce radiation induction through their respective
DNA binding sites. Thus, colony formation will not occur in
unstimulated cells.
This study also presents new findings on the
transcriptional regulation of the c-jun protooncogene in
irradiated AT fibroblasts. Cells isolated from patients with some AT
complementary groups demonstrate neither inhibition of DNA synthesis
nor G delay following exposure to ionizing radiation (27) . DNA damage-induced cell cycle arrest is proposed to be a
primary means to protect against replication of a damaged DNA template.
Genetic instability occurs in a number of disorders related to
abnormalities in p53, including Li Fraumeni syndrome and AT. In this
regard, one potential biological consequence of the lack of a G
check point in AT cells is the replication of a damaged DNA
template which may be associated with the propagation of genetic
errors(51, 52) .
We examined c-jun expression in irradiated cells, because c-jun expression
is associated with cellular proliferation. Evidence that c-jun plays a role in G/S transition is that c-jun mRNA levels peak prior to the exit of cells from
G
/G
(34, 35) . Furthermore,
microinjection of excess AP-1 binding sequences (36) or Jun
antibodies into proliferating cells prevents the onset of DNA
replication(37) . Moreover, Jun is a regulator of DNA
replication (38) and may be required for regulation of genes
that control progression through G
, such as cyclin
D1(39) . We have shown here that blocking the effects of Jun in
irradiated cells prevents the G
/S transition and enhances
cell killing by x-rays. However, the extreme radiosensitivity of AT
cells, despite prolonged immediate early gene induction and entrance
into S phase, suggests that induction of Egr-1 and c-jun is not sufficient to rescue these cells from radiation-induced
death, but is a consequence of persistent damage. Taken together with
the findings that the premitotic checkpoint and cell viability are
dependent upon the onset of S phase of the cell cycle in lower
eukaryotes(40) , the role of x-ray-mediated c-jun expression may be in the initiation of DNA replication following
genotoxic stress.
One recently proposed function of the
stress-activated protein kinases has been to activate c-jun induction following exposure to oxidative and other types of
stress(18, 19, 41, 42, 43) .
We have previously shown that x-ray-mediated microtubule-associated
protein and p90 kinase activation regulates induction of
c-jun and Egr-1(44) . We suggest that
microtubule-associated protein, pp90
, and
(MAPK/extracellular signal-regulated kinase) kinase
kinase-stress-activated protein kinase kinases may mediate the G
to S phase transition reported here. As in lower eukaryotes,
mammalian cells are likely to require independent, parallel, but
potentially interactive signaling pathways which allow for maximum
adaptability to stress(19, 41) . It is likely that the
microtubule-associated protein, pp90
and
(MAPK/extracellular signal-regulated kinase) kinase
kinase-stress-activated protein kinase signaling pathways are activated
by ionizing radiation and may act independently or interact by as yet
unknown mechanisms to control cell cycle progression and survival. Egr-1 and c-jun regulation of the onset of S phase
following x-irradiation may be due to direct interactions with genes
that govern cell cycle regulation as well as transcriptional regulation
of downstream genes that enhance cell survival.