Induction of c-fos and c-jm Gene
Expression by Phenolic Antioxidants
Hueng-Sik
Choi and David D. Moore
Department of Molecular Biology
Massachusetts
General Hospital
Boston, Massachusetts
02114
We have found that phenolic antioxidants specifically induce expression of the c-fos and c-jun protooncogenes. After treatment of quiescent human
hepatoma HepG2 cells with butylated hydroxytoluene, butylated hydroxyanisole, or other phenolic
antoxidants, the levels of c-fos and c-jun mRNAs are
substantially increased. This response is antioxidant
specific, dose dependent, and transient, with maximal levels at 3-6 h. The antioxidant-specific induction of c-fos/CAT promoter constructs in transient
transfections indicates that at least a portion of this
response is transcriptional. Deletions and point mutations map sequences required for the antioxidant
response of the c-fos promoter to the serum response element. The antioxidant-specific induction
of expression directed by a reporter plasmid containing four AP-1 sites and the induction of AP-1
DNA-binding activity confirm previous results indicating that antioxidant treatment increases AP-1 activity. (Molecular Endocrinology 7: 1596-1602,1993)
either a heterodimerof the c-fos and c-jun proteins, a
homodimerof c-jun protein, or other dimericcomplexes
that includea seriesof proteins closely related to c-fos
or c-jun (9, 10). Two recent reports demonstratethat
under certain circumstances, AP-1 sites can mediate
the response to phenolic antioxidants (11, 12). This
responseappearsto be independentof the better characterized induction by activators such as phorbol myristate, which is mediatedby protein kinase-C.
We observed that phenolic antioxidants specifically
induce expressionof the c-fos and c-jun mRNAs. The
activity of the c-fos promoter is also inducedin transient
transfections, and resultswith both deletionsand point
mutations demonstrate that the serum responseelement (SRE) is requiredfor this response.These results
suggestthat the phenolicantioxidants activate the still
poorly understoodsignaltransduction pathways associated with that complex element.
RESULTS
INTRODUCTION
Induction of c-jun and c-fos mRNA by Phenolic
Antioxidants
Butylated hydroxyanisole (BHA) and butylated hydroxytoluene (BHT) are well known antioxidants, widely
usedas preservatives of food and medicine.BHA acts
as an inhibitor of the carcinogenicityof certain xenobiotics and could be considered a likely anticarcinogen
on the basis of its activity as an antioxidant and free
radicaltrap (1). However, BHA and related compounds
have also been reported to be tumor promoters when
administeredto rodents in high doses (see Ref. 2 for
review).
Phenolicantioxidants related to BHT and BHA can
induce expression of the genes encoding NADP(H)quinone reductase (3) and the Ya-subunit of glutathione-S-transferase(4-7). Closely related compounds
that are not antioxidants do not elicit this response.
This inductionis mediatedby a short sequencetermed
the antioxidant responseelement (ARE) (8). The ARE
consensus,5’-RGTGACNNNGC-3’, is similar to the
AP-1 consensussite, 5’-TGACTCA-3’, which binds
Both antioxidants and oxidants have recently been
implicatedin direct effects on growth factor and other
signalingpathways in several recent studies(reviewed
in Ref. 13). We examineda numberof antioxidants and
related compounds for effects on the expression of
immediate-earlygenes responsiveto such signals.As
shown in Fig. 1, the levels of c-fos and c-jun mRNAs
were markedly increased by treatment of quiescent
HepG2 cells with the phenolic antioxidant BHT. This
induction was maximalat 3-6 h, much slower than the
30 min to 1 h responseof these genes to stimulation
by serum or many other factors (9, 14, 15). The responsewas not observed with proliferating cells incubated in 10% serum(data not shown).
The induction was specific to these genes.Although
someinduction was observed with the protooncogene
c-myc, BHT had no effect on the level of glyceraldehyde-3-phosphatedehydrogenasemRNA (Fig. 1) or on
the messagesof a wide variety of other genes. Those
failing to respond included other transcription factors
responsiveto various signals,such as the 8-isoforms
of the thyroid hormoneand retinoic acid receptors and
OWEWO9/93/1596-1602$03.00/O
Molewlar~tinokJgy
Copyiight
0 1993 by The Endocrine
sodety
1596
Antioxidant Induction of Protooncogenes
1597
ETOH
A
(hr)
0
6
12
BHT
24
1
3
6
12
24
C-FOS
GAPDH
C
C-FOS
,.p.
/-.
C-JUN
C-JUN
\
&Id,
BHT
c-jun mRNA (not shown).
Both c-jun and c-fos mRNAs were superinducedby
BHA
OH
the CAMP-responsivetranscription factor CREB. Other
messagesencoding proteins associatedwith signaling
processeswere also unresponsive,includingthe protooncogene c-ras and the antioncogenes~53 and retinoblastoma(Rb). The lack of responseof the antioxidant enzyme catalaseand heat shock proteins-27,-70,
and -89 indicatesthat that BHT responseis not associated with oxidant or other formsof stress(not shown).
As shown in Fig. lB, the effect of BHT was dose
dependent, reaching a maximum at 150 PM. Toxic
effects became evident at higher concentrations. The
responsewas not limited to HepG2 cells, as BHT also
induced c-fos and c-jun mRNA levels in the JEG3
humanchoriocarcinomacell line.
To gain a better understandingof the specificity of
this induction, we tested a seriesof related phenolic
compounds, includingthose shown in Fig. lC, for effects on c-fos and c-jun mFiNAs. A response was
observed only with the phenolicantioxidants, including
another commondietary preservative, BHA, as well as
catechol and hydroquinone.No effects were seeneven
with high doses of resorcinol, an isomer of catechol
that is not an antioxidant (8). Similar specificty was
observed with the antioxidant 1,2,3-trihydroxybenzene
(1,2,3-THB)and its inactive isomer1,3,5-THB, although
the level of inductionby 1,2,3-THB was lower than that
observed with other phenolicantioxidants (not shown).
However, the natural antioxidantsa-tocopherol(vitamin
E) and catechin had no effect on the levels of c-fos or
OH
OH
OH
Phenolic Antioxidants
Induce the c-fos Promoter
a Dose- and Antioxidant-Dependent
Manner
OH
CTC
the combinationof BHT and cycloheximide (data not
shown). Similar independencefrom protein synthesis
has been reported for the induction of these and other
immediate-earlygenes by many stimuli(9, 14, 15).
in
OH
HQ
OH
OH
OH
OH
1,2,3-THB
1,3,5-THB
Fig. 1. induction of c-fos and c-jun mRNAs by BHT
A, Time course. Proliferating HepGP cells were switched to
low serum (Dulbecco’s Modiied Eagle’s Medium and 1% fetal
bovine serum) and incubated for 24 h. These quiescent cells
were then treated with 100 I.LMBHT or ethanol (ETOH; 0.1%)
as vehicle control. After the indicated times, total RNA was
prepared, and 20 pg from each time point were resolved on a
1.2% agarose-formaldehyde gel and blotted to a Zeta-bind
filter. The blot was hybridized with probes from the human cfos, c-jun, and GAPDH genes; exposure times were 24, 12,
and 1 h. B, Dose dependence. HepGP cells, incubated as
described above, were treated with the indicated concentrations of BHT for 3 h. Total RNA was prepared and analyzed
Transienttransfectionswere carriedout with c-fos/CAT
reporter plasmidsto explore the mechanismof induction of fos mRNA. Initial results demonstrated that a
human c-fos promoter-chloramphenicolacetyltransferase (CAT) construct containingmore than 2 kilobases
of promter and 5’-flanking sequences was induced
approximately 2- to 4-fold by treatment with BHT,
whereasa herpesvirus thymidinekinasepromoter-CAT
construct was unresponsive.As shown in Fig. 2A, the
BHT responseof the fos promoter was comparableto
but somewhat
less than the serum response under
these conditions.
The responseof the c-fos promoter in transienttransf&ions
was similar in several respects to that of the
mRNA. As indicated in Fig. 2B, the responseof CAT
as described above. C, Antioxidant specificity. HepGP cells
were incubated as described above in the presence of ethanol
control or BHT (150 PM), BHA (300 PM), resorcinol (RSN; 200
PM), hydroquinone (HQ; 200 PM), or catechol (CTC; 200 PM).
D, Structures of the phenolic compounds used.
MOL ENDO. 1993
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Vol7 No. 12
Underthese conditions, a quite modest, but reproducible, responsewas also observed with the antioxidants
hydroquinoneand catechol (not shown). However, no
significant response was observed with the inactive
compounds1,3,5-THB and resorcinol.
The SRE Is Required for Antioxidant-Specific
Induction of the c-fos Promoter
To identify the c-fos promoter sequencesresponsible
for induction by antioxidants, a series of deletion and
point mutants was tested. As indicated in Fig. 3A, the
specific responseto BHT was lost in a deletionto -99.
Similar results were obtained in transfections treated
with 1,2,3-THB and 1,3,5THB, confirmingthe antioxidant specificity of the requirementfor these up-stream
sequences.The major site implicatedby these deletion
results is the complex element associated with responsesto serum and growth factors. A role for this
region in the antioxidant effect was confirmed by the
B
Fig. 2. Response of the c-fos Promoter to Antioxidants
A, Specific induction of the c-fos promoter by BHT or serum.
Quiescent HepGP cells were transfected with pFC4-BL, a CAT
reporter plasmid containing the c-fos promoter (34) and the
pTKGH internal control plasmid (36) as described in Materials
and Methods. Transfected cells received no additional treatment (C), or 100 PM BHT or 10% fetal bovine serum as
indicated. B, BHT dose response. The c-fos CAT reporter
pFC363 (16) was transfected as described above and treated
with indicated doses of BHT. C, Antioxidant specificity of
response of pFCCBL. The transfections described above
included the indicated phenolic compounds. Concentrations
were: BHT, 100 PM; resorcinol (RSN), 200 p~;1,2,3-THB, 200
pt.%and 1,3,5-THB, 200 PM.
over a similar range of BHT doses.
Cytotoxic effects were observed at 150 PM and higher
activity increased
concentrations
in these longer term treatments.
To avoid potential effects of toxicity, a dose of 100
PM BHT was chosen for further studies. As shown in
Fig. 2C, the pattern of responseof the c-fos promoter
to the seriesof compoundsdescribedabove generally
paralelledthat of the mRNA. Thus, treatment with the
antioxidantsBHT and 1,2,3-THB inducedCAT activity.
D
Fig. 3. The c-fos Promoter Sequences Required for Response
to Antioxidants and H202
A, 5’-Deletions. The indicated plasmids were transfected
into quiescent HepG2 cells treated with ethanol vehicle, BHT
(100 PM), or H202 (500 PM). Results represent the ratio of
treated to control (C) plates. B, Point mutants. In pTF1, an
oligonucleotide corresponding to the wild-type sequences of
the SRE and the adjacent AP-1 site inserted is upstream of
the c-fos 5’deletion to -225. The pTF2 and pTF3 contain
mutant oligonucleotides inactivating the SRE and AP-1 sites,
respectively. These plasmids have been described previously
(16). Transfections of quiescent HepG2 cells were treated with
BHT (100 PM), 1,2,3-THB (200 PM), 1,3,5-THB (200 IM), or
H202 (500 PM). In additional experiments, no antioxidant effect
was observed with the parental -225 vector.
1599
specific response of pTF1 (16) in which an oligonucleotide containing the complex SRE and the adjacent AP1 site was reinserted into a -225 c-fos promoter deletion (Fig. 38). In pTF2, the SRE was inactivated by
point mutations in the binding site for serum response
factor, whereas in pTF3, the AP-1 site was mutated.
As shown in Fig. 38, the SRE mutation blocked the
response to either BHT or 1,2,3-THB. In contrast, mutation of the AP-1 site resulted in only a modest decrease in response. We conclude that the SRE is necessary for induction of the c-fos promoter in response
to antioxidants.
BHT and other phenolic antioxidants form phenoxy
radicals as oxidation products (17). Some of the toxic
effects of very high doses of these compounds in
animals may be associated with the reaction of such
species with cellular macromolecules. Moreover, phenolic antioxidants can also generate superoxide anions
and hydogen peroxide by a process referred to as redox
cycling (18). Thus, it is possible that the induction of
expression of c-fos by the antioxidants described here
could be a paradoxical consequence of either direct
effects of the phenoxy radicals or indirect effects of
oxidative stress. The potential for such effects is increased by reports that c-fos expression is induced by
hydrogen peroxide (19, 20) and by the finding that H202
treatment can mimic antioxidants in the induction of
expression mediated by the previously described ARE
(8).
To determine whether BHT and H202 inductions of
c-fos occur by parallel or distinct mechanisms, the
series of deletion and point mutants was also tested
for response to oxidative stress. As shown in Fig. 3, A
and B, the H202 response was retained by the deletion
to -99 and by pTF2, both of which have lost antioxidant
induction. The -225 deletion alone was similarly induced by H202, but was unresponsive to antioxidants
(not shown). This disparity in the sequences required
for these effects strongly suggests that antioxidants
and oxidative stress activate the c-fos promoter by
distinct mechanisms.
AP-1 Activity Is Increased
by Phenolic Antioxidants
The response of the c-fos promoter to antioxidants in
transient transfections does not require the AP-1 site
just down-stream of the SRE (Fig. 38). However, both
previous results (4, 11, 21) and the induction of c-fos
and c-jun mRNAs described here indicate that antioxidant treatment should result in induction of AP-1 activity. As indicated in Fig. 4, expression directed by a
reporter containing four copies of an AP-1 site was
increased by addition of the phenolic antioxidants, but
not their inactive isomers, as observed with the c-fos
promoter. Expression directed by the herpes virus TK
promoter was unresponsive to all compounds in parallel
transfections, as was expression directed by a series
of TK promoter constructs with response elements for
thyroid hormone, retinoic acid, vitamin D, or estrogen
(not shown).
Fig. 4. Antioxidant Induction of AP-1 Activity
A repotter containing four tandem copies of the AP-1 site
from the vasoactive intestinal peptide (VIP) gene promoter (35)
was transfected into quiescent HepG2 cells treated with the
indicated compounds [BHT, (100 PM), resorcinol (RSN; 200
FM), 1,2,3-THB (200 PM),and 1,3,5-THB (200 PM)].C, Control.
This induction is paralleled by an increase in AP-lbinding activity. As shown in Fig. 5, nuclear extracts of
serum-starved HepG2 cells treated for increasing times
with either 1,2,3-THB or 1,3,5-THB produced several
retarded complexes in gel shift analysis with an oligonucleotide containing the c-jun AP-1 site. Over the time
course of treatment with the antioxidant, a new complex with intermediate mobility was observed. The inactive isomer had no effect. The specificity of binding
of both the antioxidant-induced and constitutive complexes was demonstrated by competition with the AP1 oligonucleotide, but not with two other unrelated
oligonucleotides. Similar results were observed with the
c-jun AP-1 oligonucleotide, using extracts prepared
from cells treated with BHT over a similar time course.
However, under these conditions no increase was
seen in specific binding to several other related oligonucleotides, including the ARE from the rat glutathioneS-transferase Ya-subunit gene. This sequence has recently been shown to specifically bind a factor(s) constitutively present in nuclear extracts from HepG2 cells
maintained in the presence of high (10%) serum concentrations (21). It is not clear whether this discrepancy
in binding results is a consequence of differences in the
growth state of the cells or of some other effect. A SRE
oligonucleotide was also used in gel shifts with these
extracts (not shown). As expected from the constitutive
binding of SRF to this element, no alteration in binding
was observed in the treated extracts.
DISCUSSION
We tested the effects of phenolic antioxidants and
related compounds on the expression of several immediate early and other genes. In quiescent HepGP and
JEG-3 cells, both c-fos and c-jun mRNAs were specifically induced by these compounds. This induction was
antioxidant specific, dose dependent, and relatively
rapid, although not as fast as the response of these
genes to growth factors and other stimuli. Thus, the
MOL ENDO. 1993
1600
Vol7No.12
A
1,2,3-THB
(hr)
0
2
5
8
1,3,5-THB
11
14
0
2
5
8
11
14
$A
previously characterized ARE (8)). It is also similar to
their response to serum stimulation of HepG2 calls (Fig.
2A) and not substantially less than their previously
reported 6- to lo-fold response to epidermal growth
factor or TPA in transient transfections of HeLa cells
(16).
The induction of c-fos and c-@ mRNAs and AP-lbinding activity reported here is consistent with a role
for these proteins in response to antioxidants. Thus, it
has recently been suggested that c-jun and c-fos are
involved
(1,2,3-THB)
-
+
-
+
-
+
Fig. 5. Antioxidant induction of AP-1 -Binding Activity
A, Nuclear extracts were prepared from quiescent HepG2
cells at the indicated times after the addition of 1,2,3-THB or
1,3,5THB and used in binding reactions with a labeled probe
consisting of the AP-1 site from the c-jun gene. B, Nuclear
extracts from either control cells or cells treated with 1,2,3THB for 11 h were incubated with the c-jun probe with no
competitor or with a lOO-fold excess of unlabeled AP-1 or
SRE competitors.
specific
results described here add phenolic antioxidants to the
large and diverse array of stimuli capable of activating
expression of mRNAs for c-fos and other immediate
early genes (see Refs. 9, 14, and 15 for review).
The antioxidant-specific
response of various c-fos
promoter constructs in transient transfections indicates
that at least a portion
of this induction
occurs
at the
transcriptional level, mediated by the SRE. However,
the apparent magnitude of the response of the c-fos
promoter to antioxidants is significantly lower than that
of the mRNA. This may indicate that additional factors,
such as mRNA stability, may contribute to mRNA induction. However, the response of the c-fos promoter
constructs to antioxidants under these conditions is
similar to the levels of induction associated with the
in the specific
activation
of expression
me-
diated by a rather complex site called the electrophile
response element (EpRE), found in the mouse glutathione-S-transferase Ya-subunit gene (4, 11). The EpRE
includes the smaller site, termed the antioxidant response element (ARE), originally identified in the rat
homolog of the same gene and in the NADP(H) quinone
reductase gene (8). A portion of the ARE consensus
sequence (5’-ggTGACaaaGC-3’) is similar to the AP-1
consensus (5’-TGACTCA-3”). Moreover, the EpRE has
been reported to be bound by the combination of c-jun
and c-fos (ll), whereas the ARE can be bound by
purified c-jun (8). Under at least some conditions, consensus AP-1 sites can confer response to antioxidants
in transient transfections (11) (Fig. 4).
The response to phenolic antioxidants clearly involves much more than simply activation of expression
of c-fos and c-jun, however. Thus, analysis of the
specific binding of nuclear proteins to the ARE directly
implicates additional factors (21). These results showed
that the AP-1-like element in the ARE core is essential
for such binding in an electrophoretic mobility shift
assay, but the mobility of the complex was distinct from
that produced by purified c-jun. In addition, a large
excess of a consensus AP-1 site did not compete for
formation of the nuclear extract complex. It was concluded that the gel shift complex does not consist of
jun homodimers or jun-fos heterodimers. The studies
reported here implicate additional factors in the antioxidant response. The induction of AP-l-binding activity
and function is rather modest by comparison to the
level of induction of the mRNAs. Moreover, the mapping
results demonstrate that the c-fos promoter requires
the SRE, but not the adjacent AP-1 site, for antioxidantinduction.
The SRE was also identified
as a
target for activation of fos expression in response to
the antioxidant pyrrolidine dithiocarbamate by Meyer et
a/. (22) in experiments reported after this work was
completed. Although the lack of involvement
of the
nonconsensus
c-fos AP-1 site in the antioxidant response may seem inconsistent with the response of
the 4xAP-1 reporter, it is consistent with previous studies indicating that the c-fos AP-1 element is not associated with autoactivation
of expression, in contrast to
the positive feedback effects observed with the AP-1
site in the c-jun promoter, for example (23). It remains
that the c-fos element could show antioxidant
responsiveness under other circumstances, however.
The basis for the activation of the SRE by antioxipossible
dants
is unknown.
In general,
of course,
the overall
Antioxidant Induction of Protooncogenes
redox state of the cell must be maintainedat a stronalv
reducing level to prevent undesirableeffects, such&
the oxidation of free thiol groups necessary for the
activity of numerousenzymes. However, variations in
redox parametershave been directly implicatedin controlling the activity of several transcription factors, includingjun and fos (24-26) as well as NFKB (27, 26)
and the glucocorticoid receptor (29). The recent report
that the solubleantioxidant N-acetylcysteine can inhibit
a tyrosine kinasedependent induction of c-jun expressionsuggestsan indirectrole for redox effects in growth
factor signaltransduction pathways (30). Thus, one or
more of the complex group of proteins that interact
with the SRE (31-33) could either be directly altered by
a redox effect or be activated indirectly as a consequenceof effects of the antioxidants on a signaltransduction pathway.
Responsesto antioxidants may also be associated
with the oxidative stress that can result from redox
cycling (6, 16). The lack of effect of the phenolicantioxidants on expressionof the heat shock protein-27, -70,
or -89 or catalasemRNAs arguesagainsta role for this
process in the effects described here, because all of
these genes are induced by oxidative stress. Further
evidence excluding a role for oxidative stress is provided by mappingof the antioxidant and H202effects
on the human c-fos promoter to distinct regulatory
elements.However, this result is in contrast to a recent
report implicatingboth the SRE and AP-1 sites of the
mouse c-fos oromoter in resoonse to H202 in stablv
transfected mouse epidermal’cells (19). Although the
basisfor this discrepancy is uncertain, it could involve
differencesin cell lines,transfectiontechniques,or other
issues.
Finally, the induction of protooncogene expression
by BHT and BHA may be relevant to reports that high
levels of these widely used antioxidants exhibit tumor
promoteractivity (2). Although our resultsare obviously
far from establishinga direct connection between the
inductionof protooncogeneexpressionand such activity, they provide a specificdirection for further analysis
in this area.
MATERIALS AND METHODS
Plasmids
c-&s CAT reporter plasmids pFC1 -BL and pFC4-BL contain
approximately 2250 and 404 basepairs of the human c-fos
promoter, as previously described (34). The CAT reporters
pFC363, pFC225, andpFC99,containing
theindicatedamount
of human c-fos promoter sequence, and pTF1, pTF2 and
pTF2, in which oligonucleotides corresponding to c-fos sequences from -316 to -264 are inserted upstream of the
-225 deletion, have also been described (16). The pTF1
contains wild-type c-fos sequence, pTF2 has four point mutations that inactivate the SRE-binding site, and pTF2 has two
point mutations that inactivate the adjacent APl site. The
4xAP-1 CAT contains four tandem copies of the AP-1 site
from the vasoactive intestinal peptide gene promoter, as previously described (35). The human (h) GH expression vector
pTKGH was described in an earlier report (36).
1601
Cell Culture and Chemicals
HepGP and JEG3 cells were grown in Dulbecco’s Modified
Eagle’s Medium supplemented with glutamine and 10% fetal
calf serum. Before treatments with various compounds, cells
at 40% confluence were shifted to medium containing 0.5%
fetal calf serum for 24-46 h. BHT, BHA, resorcinol, catechol,
hydroquinone, 1,2,3-THB, and 1,3,5-THB were obtained from
Sigma (St. Louis, MO) and dissolved in 100% ethanol. The
cytotoxicity of these compounds was monitored by counting
trypan blue-stained cells. Cells were transfer&d at 60% confluence using CaPO, precipitation with 3 Ag of the various
CAT-expressing plasmids and 4 Ag of the hGHexpressing
control plasmid pTKGH (36). Cells were incubated for 6 h with
the DNA precipitate, washed, and serum starved (0.5% serum)
for 24 h before treatment with various compounds. CAT
activity was assayed using phase extraction (37) and normalized to levels of hGH expressed by control plasmids, as
previously described (36). All results represent the average of
at leasttwo independently
transfectedplates,normalizedto
hGH. Error bars indicate the calculated SOS. All figures represent data from single experiments, with at least two independently transfected plates for each condition. Consistent results
were obtained from independent experiments.
Northern Blot Analysis
Total RNA was isolated using guanidinium isothyocyanate and
CsCl density gradient centrifugation (37). Twenty micrograms
of total RNA were resolved by electrophoresis on a 1.2%
agarose gel containing formaldehyde and transferred to a
nylon membrane (Zeta-Probe, BieRad, Richmond, CA). Human c-fas, cjun, c-myc, mouse glyceraldehydephosphate dehydrogenase, and additional probes were labeled to high spscific activity using random priming, and a total of 2-4 x lo6
cpm was used for hybridizations. Blots were washed twice for
5. min at room temperature in 2 x SSC and 0.1% sodium
dodecyl sulfate (SDS), followed by 1 h at 65 C in 0.5 x SSC
and 0.1% SDS. For rehybridization,blotswere strippedby
boiling in 0.1 x SSC and 0.5% SDS twice for 20 min.
Gel Shift Assay
Nuclear extracts were prepared as previously described (37).
Binding reactions contained 7.5 Pg nuclear extract protein, 0.5
ng =P-labeled oligonucleotide, and 1 pg poly(dldC&oly(dldC) in a buffer containing 10 mM Tris (pH 7.6) 50 mM NaCI, 1
mM EDTA, 0.5 mM dithiothreitol, and 5% glycerol, as previously
described (39). Reactions were incubated for 20 min at room
temperature, and bound and free probes were resolved by
electrophoresis on a 5% polyacrylamide gel in 40 mM Trisacetate and 2 mM EDTA (37). The c-jun AP-1 oligonucleotide
sequence was 5’-GATCCTTGGGGTGACATCATGGGCT-3’.
and the c-fos SRE oligonucleotide sequence was 5’GATCCAGGATGTCCATAlTAGGACATCTGT-3’.
Acknowledgments
We thank Drs. Ronald Prywes for numerous human c-fos
promoter-CAT promoter constructs, Alessandro Weisz for
pFCl-BL and pFC4-BL, Steven Fink for the 4xAP-1CAT re
porter plasmid, Sam Lee for c-fos and c-jun cDNA probes, and
Michael Greenberg for helpful discussions.
Received May 7,1993. Revision received August 27,1993.
Rerevision received September 26, 1993. Accepted September 30,1993.
Address requests for reprints to: Dr. David Moore, Molecular Biology, Massachusetts General Hospital, Wellman - 9th
Floor, 50 Blossom Street, Boston, Massachusetts 02114.
This work was supported by a grant from Hoechst AG.
Vol7No.12
MOL ENDO. 1993
1602
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