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[CANCERRESEARCH 57, 3569—3576,August 15, 19971
ABSTRACT
Cross-coupling of active protein-i (AP-1) and nuclear factor (NF)-icB
has been reported. In the present study, we investigated the possibility
that both ofthese two transcription factors might contrIbuteto the process
oftumor promoter-induced transformation. To establish a stable reporter
cell system, two reporter genes were stably transfected into a JB6 mouse
tumor promotion-sensitive (P+) cell line: a luclferase reporter controlled
by a collagenase AP-1 sequence and a chloramphenlcol acetyltransferase
reporter controlled by an interleukin 6 NF-icB sequence. This double
reporter cell line maintained the phenotype oftumor promotion sensitivity
and was able to report basal or induced AP-1 and NF-scB transactivation.
The cytokine tumor promoter tumor necrosis factor (TNF)-a transacti
vated NF-.cB and AP-1for both DNAbinding and transcriptional activity.
Pyrrolidine dithiocarbamate, an antioxidant that acts as an NF-.cB inhib
itor, efficiently inhibited 12-O-tetradecanoylphorbol-13-acetate (TPA) or
TNF-a induced NF-scB as well as AP-1 transactivatlon and cell transfor
mation, suggesting dependency of transformation on both transcription
factors. The AP-1 transrepressing-retinoid SR11302 transrepressed AP-1
and cell transformation when these were TPA induced but not when
TNF-a induced, indicating different signaling pathways for TNF-a and
TPA. Supershift electrophoresis mobility shift assay revealed that Jun B
and c-Jun were absent from the AP-1IDNA complex following TNF-a but
present following TPA treatment. Together, these results suggest that both
AP-1 and NF-.cB activation may be required for transformation whether
Induced by TPA or by TNF, and the differential sensitivity of TPA and
TNF-a-lnduced transformation to Inhibition by a retinoid might be cx
plalned by differences In the composition of the DNA-bound AP-1 corn
plexes.
INTRODUCTION
The JB6 family of mouse epidermal clonal genetic variants that are
transformation sensitive (P+) or resistant (P—)provides a suitable in
vitro model for studying critical gene regulation events that occur
during carcinogenesis (1—3).Studies with both gene and small mol
ecule inhibitors have provided evidence that AP-l transactivation is
required for tumor promoter-induced cell transformation. Elevated
c-Jun expression and AP- 1 transactivation are induced by tumor
promoters TPA3 or epidermal growth factor in promotion-sensitive
but not in promotion-resistant cells (4). TPA-induced cell transforma
tion is blocked by AP- 1 inhibitors of several classes including reti
noids, glucocorticoids, and the dominant-negative c-Jun TAM67 (5),
which sequesters endogenous Jun and Fos in transcriptionally inactive
complexes (6). Recently, we have found that transactivation of NF-,cB
as well as of AP- I occurred during progression in a mouse keratino
Received3/4/97;accepted6/I3/97.
Thecosts of publicationof thisarticleweredefrayedin part bythe paymentof page
charges.Thisarticlemustthereforebeherebymarkedadvertisementinaccordancewith
18 U.S.C. Section 1734 solely to indicate this fact.
I Supported by NIH Howard Hughes Medical Institute Research Program (to C. W.).
2 To whom requests for reprints should be addressed, at Gene Regulation Section,
Laboratory of Biochemical Physiology, Frederick Cancer Research and Development
Center,P.0. BoxB,Building560,NationalCancerInstitute/NIH,Frederick,MD21701-
1201. Phone: (301)846-1333; Fax: (301) 846-6143; E-mail: lij@ncifcrf.gov.
3 The abbreviations used are: TPA, l2-O-tetradecanoylphorbol-l3-acetate; NF-,cB,
nuclearfactor-KB:AP-l, activeproteinI; RA,trans-retinoicacid;RAR,retinoicacid
receptor; PDTC, pyrrolidine dithiocarbarnate; CAT, chloramphcnicol acetyltransferase;
IL, interleukin; CMV, cytomegalovirus; EMEM, Eagle's MEM; PBS, fetal bovine serum;
TNF, tumor necrosis factor; EMSA, electrophoresis mobility shift assay.
cyte (7) and in a human keratinocyte progression model (8) and that
TAM67 inhibited activation of both transcription factors. These find
ings raise the possibility that NF-KB activation could be required
instead of or in addition to AP-l activation. We thus have investigated
the significance of NF-KB transactivation and the possible interaction
between the two transcription factors in the process of neoplastic
transformation.
Both AP-l and NF-KB are heterodimeric nuclear transcription
factors. AP-l consists of Jun/Jun homodimers or heterodimers of Jun
(c-Jun. Jun B, Jun D) and Fos (c-Fos, Fos B, Fra-l, and Fra-2; Ref. 9).
Many stimuli including tumor promoters regulate AP-l protein bind
ing to the consensus AP-1 binding sequence on transcriptional pro
moters and stimulate gene transcription. Some of these AP- 1-regu
lated gene transcripts may mediate neoplastic transformation (4, 10,
11). NF-KB complexes also consist of members of a multigene family
comprised of five major proteins: p50, p65(Rel A), c-Rel, p52, and
Rd B (12, 13). In general, the most abundant dimer of the inducible
NF-KB is a p5O/p6S heterodimer. Both p65 and p50 contribute to
DNA binding; only p65 transactivates (14). NF-KB activity is regu
lated by a binding protein 1KB in the cytoplasm. Under unstimulated
conditions, NF-KB is quiescent in combination with 1KB and is
activated for nuclear entry when dissociated from 1KB in response to
viruses, bacteria, radiation, oxidants, and cytokines such as TNF-a
(15). Several1KBproteinshavebeencharacterized(I2, 16—18).
Disruption of 1KB by overexpressing antisense RNA activated NF-KB
binding activity and induced cell transformation ( 19). Interestingly,
cross-interaction of c-Jun or c-Fos but not Jun D or Jun B with the
NF-KB subunit p65 has been reported to lead to a synergized potential
of both AP-l and NF-KB transactivation (20).
Several inhibitors of AP-l or NF-KB transactivation are known.
Natural RA not only inhibits AP-l but also prevents phorbol ester
induced transformation of mouse JB6 cells (21) and initiated mouse
skin (22) and suppresses tumor phenotype in JB6-derived tumor cells
(23, 39) as well as other tumor cell lines (24, 25). LigandedRARs
prevent AP-IIDNA binding (26) by a mechanism postulated to in
volve direct interaction with c-Jun akin to the c-Jun/glucocorticoid
receptor interaction (27). Although the DNA-binding domain of
RARs is required, the RA-RAR does not bind to the AP-l site (28,
29). Several new synthetic retinoids have been shown to selectively
inhibit AP-l transactivation and others only to induce RA response
element transactivation (29, 30). Only the AP- 1-repressing retinoids,
such as SR1 1302, suppressed AP-l transactivation and cell transfor
mation in JB6 cells (23). Likewise, other compounds block NF-KB
transactivation. NF-KB behaves as an oxidative-stress responsive fac
tor that can be directly activated by hydrogen peroxide (3 1, 32) and
repressed by antioxidant compounds (32, 33). PDTC, a proven free
radical scavenger, efficiently inhibits NF-KB transactivation (34, 35),
apparently by acting upsteam of 1KB dissociation with a resulting
block in NF-KB transport to the nucleus and binding to DNA (33).
PDTC also prevents NF-icB-dependent gene expression (36).
To further probe the roles of AP-l and NF-scB and a possible link
between the two transcription factors in tumor promoter-induced
neoplastic transformation, we have investigated the two factors in a
double-reporter JB6 P+ cell line, which has been stably cotransfected
3569
Inhibitors of Both Nuclear Factor-i@B and Activator Protein-i Activation Block the
Neoplastic Transformation Response1
Jian-Jian Li,2 Chandra Westergaard, Paritosh Ghosh, and Nancy H. Colburn
Gene Regulation Section, Laboratory of Biochemical Physiology fJ-J. L, C. W., N. H. CI, and Laboratory of Experimental Immunology (P. G.J. National Cancer Institute,
Frederick Cancer Research and Development Center, NiH, Frederick, Maryland 21702
AP-I AND NF-KB IN TRANSFORMATION
with AP-l and NF-KB reporter genes. The results suggest that both
AP-l and NF-,cB activation may be involved in tumor promoter
induced transformation, with NF-KB activation occurring first. Sig
naling for AP-l transactivation induced by TNF-cx differs from that
induced by the phorbol ester TPA by an apparent bypass of c-Jun in
the TNF-a-induced AP-l complex.
MATERIALS AND METHODS
Reagents. TPA was purchased from Chemicals for Cancer Research
(Edina, MN) and DMSO from Pierce (Rocklord, IL); murine TNF-a was from
Peprolech, Inc. (Rocky Hill, NJ). Trans-retinoic acid was from Sigma Chem
ical Co. (St. Louis, MO), and the synthetic retinoids were the same as reported
(23,30)andkindlyprovidedbyDr.M.I. Dawson(SRIInternational,Menlo
Park, CA). LipofectAMlNE was from Life Technologies, Inc. (Grand Island,
NY); [a-32P]ATP was from Amersham Life Science Co. (Arlington Heights,
IL). Luciferase assay substrate was from Promega Corp. (Madison, WI). G418
sulfate was purchased from Life Technologies, Inc. (Gaithersburg, MD), and
hygromycin B was from Calbiochem-Novabiochem Co. (Center Court, San
Diego, CA). The antibodies against c-Jun, Jun B, and Jun D were obtained
from Santa Cruz Biotechnology Co.
Plasmids. A sequence of the collagenase promoter region (—73 to +67)
containing one AP-l binding site (37, 38) was excised from the collagenase
AP-l CAT construct and inserted into a luciferase reporter vector p012-basic
(Promega) to make the AP-l luciferase reporter construct as reported (39).
IL6-NF-KBCAT reporter plasmid constructed by inserting the IL-6 promoter
region (—194 to —I 14 containing four NF-KB binding sites and no AP- 1 sites)
into pBLCAT2 reporter was kindly provided by Dr. H. Young at National
Cancer Institute-Frederick Cancer Research and Development Center, NIH.
CMV-neo-selection plasmid (pBKCMV) and hygromycin-selection plasmid
(pCEP4) were obtained from Invitrogen Co. (San Diego, CA). CMV-j3-
galactosida.se plasmid was purchased from Clontech Laboratories, Inc. (Palo
Alto, CA).
Establishrnent of the JB6/AP/icB Reporter Cell Line. Two-step reporter
gene cotransfection was performed to generate JB6 cell lines that have inte
grated AP-i and NF-KB response elements driving luciferase and the CAT
gene, respectively. Briefly, a JB6 P+ cell line Coll9, which had been cotrans
fected previously with collagenase-luciferase reporter and pBKCMV-neo gene
(23), was transfected with the NF-KB CAT reporter and hygromycin-selecting
plasmid pCEP4. Twenty-five individual clones were selected by ring isolation
and passaged in 4% FBS EMEM containing hygromycin (250—500 @g/ml).At
passage eight, three clones maintaining CAT activity were cultivated for
additional passages. One clone Coll9Nl , which maintained a consistent CAT
and luciferase activity for more than 10 passages, was chosen for the two
transcription factor study and was designated as the JB6/AP/KB reporter cell
line. The JB6/AP/KB cell line was grown in 4% FBS EMEM supplemented
with G418 (200 @g/ml)and hygromycin (250 ,@g/ml)for 8—10passages and
the same medium without selection antibiotics for three more passages before
experiments. The basal luciferase and CAT activities in the established cell
lines were tested every three to five passages. The transcription factor activa
Lion was measured by testing luciferase or CAT activity following stimulation
by TPA or TNF-a. The response to TPA-induced transformation was tested by
anchorage-independent growth in soft agar as described (1, 2) and compared to
the parental JB6 P+ cells.
AP-1 or NF-scBTransactivation by Tumor Promoters and Inhibition by
Antitumor Promoters. JB6/APIkB reporter cells were plated 3 X l0@cells/
well in a 24-well dish 24 h before each experiment. For transformation
experiments, cell culture medium was replaced at different time intervals with
2% EMEM containing TPA or TNF-a, with or without antitumor promoters
SRI 1302 (l0@—l0@M)or PDTC (10—40,.@M).Cell lysates were prepared,
and induced AP-l or NF-,cBtransactivation was determined by assaying for
luciferase activity or CAT activity, respectively, from the same samples when
treatments shown were identical.
Luciferase Assay for AP-1-dependent Transactivation. For studying
AP-1 transactivation, JB6/AP/,cB cells were plated in a 12-well cell culture
plate and cultured in 4% EMEM at 36°Cfor 12—24h (70—80%confluence)
before experiments. For tumor promoter treatment, cells were then cultured in
EMEM with 2% FBS and with or without TPA (10 ng/ml) or TNF-a (200
units/mI) and with or without transcription factor inhibitor retinoid SR11302
(l0@ M—105M)or PDTC (10—40 @M)for different lengths of time. After
treatment, cells were washed by PBS and lysed with the luciferase assay cell
lysis buffer (Promega). The cell lysate was then mixed with an equal amount
of luciferase assay reagent (Promega), and luciferase activity was measured by
a luminometer (Monolight 2010; Analytical Luminescence Laboratory, San
Diego, CA). Results wereexpressed as ratioor fold induction of the activityof
cells with or without tumor promoter treatment. When treatments shown were
identical, the same samples were used to measure both transcription factors.
CAT Assay of NF-icB-dependentTransactivation. Cells were collected
following the same treatments as used in the luciferase activity assay. Cell lysis
used a freeze and thaw method, and the lysates were heat treated for 10mm at
60°C.CAT enzyme activity was assayed according to the published method
(40) using 5—10@Lgof cell lysis protein in a reaction volume of 110 p1
incubated at 37°Cfor 2—3h or at room temperature for overnight. Reaction
products were analyzed by TLC followed by autoradiography and normalized
for protein recovery. Alternatively, CAT activity was measured by the CAT
ELISA method by using the CAT-ELISA measuring kit (Promega). Quantita
tion of CAT enzyme activity was calculated by radioactivity scanner or by
ELISA plate reader. Results were expressed as the ratio of activity with or
without tumor promoter treatment, or fold induction.
Nuclear Protein Preparation. Nuclear protein was prepared according to
the published method with some modification (41). Cells (5 X l0@)were
collected by trypsinization and washed three times by PBS and resuspended in
1 ml of cell lysis buffer containing 50 mt@iKCL, 0.5% NP4O,25 mai HEPES
(pH 7.8), 2 mMPMSF, 2 pg/ml leupeptin, 4 pg/ml aprotinin, and 100 pM
DTT. The cell lysis mixture was centrifuged at 4°Cfor 1 mm, and the pellets
were washed with 0.3—0.5ml washing buffer once by centrifuging at 4°Cfor
1 mm. The cell nuclei were then resuspended in 50—100p1 of nuclear protein
extraction buffer containing 500 mM KC1, 25 mist HEPES (pH 7.8), 10%
glycerol, I mM PMSF, 1 pg/mI leupeptin, 2 pg/mI aprotinin, and 100 pM Dli'.
The nuclear extraction was centrifuged at 4°Cfor 10 mm, and the supematants
were saved as nuclear protein extract and stored at —70°C.The protein
concentration of the nuclear extract was measured before EMSA analysis.
EMSA Analysis. Mobility shift assays were performedto detect AP-1 or
NF-KB DNA binding activity after exposure to the tumor promoter TPA or
TNF-a for the indicated times. An AP-l binding sequence from the human
collagenase promoter region, 5'-AGCATGAGTCAGACACCFCTGGC-3'
[human collagenase AP-1, position —73to —54](37), and NF-icB oligonu
cleotide 5'-AG1i'GAGGGGACTIi'CCCAGGC-3' from Promega were end
labeled with [32PIdATP. Nuclear proteins (extracted from the reporter cells and
equalized at 3—5pg) were added to the DNA binding buffer, which contained
1.5 pg of poly(dIdC), 1 M Tris (pH 7.5), 2 MKC1, 10% Ficoll, 0.5 M EDTA,
and 1MDli'. The reaction mixture was incubated on ice for 10 mm and then
further incubated for 20 mm at room temperature after adding S X l0'@cpm
32P-labeled oligonucleotide probes. The DNA-protein complexes were re
solved in a 6% nondenaturing acrylamide gel and electrophoresed for 1.5—2h
atroomtemperature.The gel was driedandexposed to Kodakfilmat —70°C
for overnight.
Supershift Assay. For supershift assay, the nuclear extracts were first
incubated with antiserum to AP-l or NF-KBsubunits of various concentrations
at room temperature for 20 mm and then further incubated with 32P-labeled
DNA oligonucleotide for another 20 mm. The supershift complexes were
resolved in a 6% nondenatunng acrylamide gel and electrophoresed at room
temperature as described above.
Anchorage-independent Transformation. TPA- or TNF-a-induced cell
transformation was assayed in the JB6/AP/KB reporter cells by using the
described induction of anchorage-independent growth shown to be irreversible
and to correlate with tumorigenicity (1, 2). Briefly, exponentially growing
JB6/AP/scBreporter cells were washed three times with PBS and trypsinized
with 0.033% trypsin and 0.13 mist EDTA, diluted and suspended in 1.5 ml
0.33% agar EMEM over 7 ml of 0.5% agar medium containing TPA (3 ng/mI,
5 flM) or TNF-a (200 units/ml) with or without AP-l or NF-KB inhibitor
RAll3O2 or PDTC, respectively. The cultures were maintained in a 36°C
incubator for 14 days, and the anchorage-independent colonies greater than
eight cells were scored by a computerized image analyzer. The efficiency of
inhibition of TPA- or TNF-a-induced cell transformation is presented as
percentage of the transformation frequency when the cells were treated with
TPA or TNF-a only.
3570
AP-l AND NF-acBIN TRANSFORMATION
Rapid Induction by TNF-a and Slower Induction by TPA of
Both NF-,cB and AP-1 Transactivation. In the present study, TPA
and TNF-a, both promoters of neoplastic transformation in JB6 P+
cells, were compared for their ability to activate transcription factors.
Fig. 2A confirms and extends previous studies to now show that
activation of AP-l occurs following TNF-a as well as after TPA
exposure. Maximal AP-l activation by TNF-a was 2-fold at 3 h,
whereas that by TPA was about 3-fold at 16 h but measurable much
earlier. Maximal AP-l-DNA binding (Fig. 2B) occurred at 12 h
following TPA, thus paralleling the transcriptional activation. The
time course of AP-I1DNA binding activity induced by TNF-cs differed
from that induced by TPA (Fig. 2C). Increased binding activity was
detected as early as 1 h, and the maximal level occurred at 2 h
following TNF-cx exposure (Fig. 2C). Fig. 3A shows a similar maxi
mal 1.8-fold transactivation response for NF-.cB following TPA or
TNF-a exposure. Maximal TPA-induced NF-KB transactivation was
sustained from 8 to 24 h, whereas that induced by TNF-a occurred
rapidly (less than 2 h) and was transient (Fig. 3A). Transactivation by
TPA of NF-KB was half maximal at less than 2 h, whereas AP-l
induction was half-maximal at 6 h. TPA-induced NF-scB-DNA bind
ing paralleled TPA-induced NF-KB transactivation (Fig. 3, A and B).
Likewise, NF-scB DNA binding activity was increased within 1 h
following exposure to TNF-a (Fig. 3C). In all cases, the fold-increase
of DNA binding activity of both AP-l and NF-KB was higher than the
fold-induction of transcriptional activity. In summary, TNF-a induced
early and transient transactivation of both AP-l and NF-icB, whereas
TPA induced later but more sustained transactivation of both iran
scription factors. For TPA and possibly for TNF-a, NF-scB transac
tivation preceded AP- 1 transactivation.
PDTC, a Reported NF-scB Inhibitor, Also Inhibits AP-1 Trans
activation. Because tumor promoter-induced activation of NF-KB
appears to occur more rapidly than AP-l activation, the possibility
arises that the activation of NF-.cB causes AP-l activation. If this is
the case, we would predict that inhibitors of NF-KB would inhibit
AP-l transactivation in JB6 cells, whereas AP-l inhibitors may or
may not inhibit NF-KB activation. PDTC, an effective antioxidant, is
a potent inhibitor of NF-KB transactivation (33—35).PDTC has been
reported to efficiently inhibit NF-KB but not AP-l transactivation
(33). PDTCwas thus used as an inhibitorto block NF-KBtransacti
vation following exposure to tumor promoters TNF-a and TPA. As
expected, Fig. 4 shows that PDTC efficiently inhibited NF-KB trans
activation induced by TPA or TNF-a in a dose-dependent pattern
(Fig. 4B). Consistent with our hypothesis, PDTC also inhibited AP-l
@, transactivation in JB6 cells (Fig. 4A). Similar inhibition by PDTC of
both transcription factors was seen after exposure to epidermal growth
factor, another transformation promoter in the JB6 model (data not
shown). This inhibition of AP-l and NF-KB was relatively specific
because no inhibition of p53-dependent transcription occurred in
response to PDTC, and the dose range used in the present experiments
was not growth inhibitory in JB6 cells (data not shown). These results
are compatible with the possibility that NF-scB activation by TPA or
TNF-a is an early event leading to AP-l transactivation. The alter
native possibility that PDTC inhibits NF-KB and AP-l separately,
interacting directly with AP-l proteins in JB6 cells, cannot, however,
be excluded.
The AP-1-repressing Retinoid SR11302 Inhibited TPA but not
TNF-a-induced AP-1 and NF-icB Transactivation. To specifically
inhibit AP-l activation, we used an AP-l repressing but not RA
response element-transactivating retinoid, SRI 1302 (23, 30). RARs
when liganded to the AP-l transrepressing retinoids inhibit AP-l
DNA binding and transactivation by a mechanism postulated to in
volve RAR/c-Jun interaction (26, 28, 29). Fig. 5 shows differential
transrepression by retinoid at the times of maximal induction, 18 h
—0——Luclferase Activity (log)
...-@ CAT ActIvity
5
4
3
2
1
0-
Coll9 cell line
transfection
with NF-kB reporter
N
.@
V
5
4
3
2
1
0
0 10 20 30
Passage Number
Fig. 1. Stable expression of both AP-l luciferase and NF-scB-CAT in the double
reporter 186 cell line. The JB6/AP/.cB reporter cell line was established by transfecting
IL-6-NF-.cB reporter plasmid into the Col-AP-l reporter cell line Co119 (23) and selected
in hygromycin as described in “Materialsand Methods.― Of twenty-five individual
drug-resistant clones, one designated as the JB6/AP/scBcell line was chosen for further
studies based on retention of promotion-sensitive (P+) phenotype and reproducibly
detectable basal expression of reporter activity. Both AP-l and NF-KBbasal activities are
shown for passages 8—24.The AP-l luciferase activity is presented as log conversion of
theactualreading,andNF-icBCATactivityisnormalizedtothevalueforthefirstpassage
following selection.
3571
RESULTS
Establishment of AP-1 and NF-scB Reporter Transfectant JB6
p+ Cells. To characterize both AP-l and NF-KB transactivation
responses in the same cells in which neoplastic transformation out
comes could be assessed, we stably transfected JB6 promotion-sen
sitive (P+) cells with AP-l and NF-#cB gene reporters. These included
a collagenase-luciferase AP-l reporter containing one AP-l binding
site and an IL-6 NF-KB CAT reporter containing four NF-KB binding
sites. These reporters have been shown to respond to inducers of AP-l
or NF-scB transactivation and not to “report―activation by other
transcription factors (Ref. 23 and data not shown). The use of stable
reporter transfectants offers the additional advantages of reducing the
variability generated from cellular trauma during transient transfec
tion and of reflecting endogenous transcription of genes within chro
matin. The previously described AP-1 reporter cell line derived from
the JB6 parental Cl4l P+ cells (23) was used as recipient for the
NF-KB reporter introduced in the present study. The JB6/AP/icB
reporter cell line was established by transfecting the IL-6-NF-KB CAT
reporter plasmid into this AP-l reporter cell line and selecting and
monitoring for multiple passages. To have a similar sensitivity for
measuring both reporter responses, we chose a cell line in which basal
luciferase activity (AP-l) and basal CAT activity (NF-icB) were
consistently detectable after IL-6-NF-scB reporter transfection. As
indicated in Fig. 1, basal AP-l and NF-KB activities were detected in
the JB6/AP/scB reporter cell line consistently and simultaneously, thus
providing a suitable tool for measuring fold induction of both tran
scription factor activities. To ensure that the original transformation
sensitivity in the reporter gene transfectants was not altered by the
process of gene transfection and selection, the anchorage-independent
transformation response of JB6/AP/icB cells to tumor promoter TPA
or TNF-a was tested (2, 23,42). The JB6/AP/.cBcell line retaineda
similar sensitivity to the TPA- or TNF-a induced cell transformation
that characterizes the parental P+ Cl4l cells (Ref. 43 and data not
shown).
U I 2 4 S !2 24 IS 1
AP-l AND@ IN TRANSFORMATION
Time (hours)
I—i— —@
() 1 2 (2 24 3(
Time (hour)
Time (hours)
B
Time(hour)
I 6.6 6.4 3.6 5.8 3.3 0.7
A—0— TPA
C --..— TNF
@ —0-@- control
TNF-a 0 1 2 4 8 12 24 36 F
(long/mi) Time(hour)
Fig. 2. AP-l transactivation and DNA binding activity induced by TPA or TNF-a. A,
AP-l transactivation following TPA or TNF-a treatment. Thirty thousand JB6/AP/KB
cells growing in 12-well tissue culture plates were exposed to tumor promoter TPA (10
ng/ml) or TNF-a (10 ng/ml) in EMEM with 2% FBS for the time intervals indicated. Cell
lysates from duplicate wells were prepared. and luciferase activity was measured by
adding100@zlof luciferasereactionsolutionto 100@.dofcelllysate(variationis lessthan
10%). Data are presented as fold induction normalized to the luciferase activity in control
wells (cultured in the same medium without TPA or TNF-a treatment) at the correspond
ing time points. B and C. EMSA of AP-l binding activity in JB6/AP/.cB reporter cell line
following TPA (B) or TNF-a (C) treatment. JB6/AP/KB cells were plated in 100-mm cell
culture dish (5 X 106cells/dish) in EMEM with 4% FBS and culwred for 12—16h (80%
confluence). The cell medium was replaced by EMEM with 2% FBS and with tumor
promoterTPA(10ng/ml)orTNF-a(10ng/ml)andculturedfordifferenttimeintervals
as indicated in the transactivation assay described in A. For EMSA, 3 pg of nuclear
protein purified from cells at each time point were incubated with 3 X 10' cpm
32P-labeledcollagenaseAP-l oligonucleotideandseparatedby6%acrylamidegel elec
trophoresis as described in “Materialsand Methods.― The specificity of binding was
verified by using bOX excess of cold collagenase oligonucleotide and for nonspecific
binding using SP-l oligonucleotide (data not shown). Quantitation of DNA-binding
activity was estimated by radioactivity scanner andIor densitometry anu normaiizeu to LOC
value for the control cells (top). F, free probe control. An additional EMSA experiment
using separate nuclear extracts gase similar results.
3572
A
0
:: —‘O—j@p@
.@ —.—- TNF
@ —@0—-control
S
I-
4
z
I 0.9 0.8 1.6 3.2 3.9 3.8 2.4
@:
NS -÷
TPA
(lOng/mi)
C
NF-icB -+
NF-icB-+
NS -+
TNF-a
(lOng/mi)
Fig. 3. NF-scBtranscription and DNA binding activity induced by TPA or TNF-a. A.
NF-KB transactivation following TPA or TNF-a treatment. Tumor promoter treatment
was identical to that used for measuring AP-l luciferase activity (Fig. 2A), and CAT
activity was measured by TLC or CAT-ELISA methods as described in “Materialsand
Methods―(variationislessthan 10%).Dataarepresentedas foldinductionnormalizedto
theCATactivityincontrolwells(culturedinthesamemediumwithoutTPAor TNF-a
treatment) at the corresponding time points. B and C, EMSA of NF-scBbinding activity
in JB6/AP/,cB reporter cell line following TPA (B) or TNF-a (C) treatment. The same
nuclear extracts used for AP-l binding (Fig. 2, B and C) were incubated with 3 X 10―cpm
32P-labeled NF-,cB oligonucleotide for gel shift assay. Specificity of binding was verified
by using IOOXexcess of cold NF-KB oligonucleotides and for nonspecific binding using
SF-I u1i@oiiu@ieoti&@,(d@Stanotshown).Quantitationof NF-.cBDNAbindingactivitywas
the same as described in Fig. 2 and normalized to the value for untreated control cells
(top). F, free probe control. Similar results were obtained using duplicate nuclear extracts.
I 1.2 1.1 1.4 1.9 3.9 4.2 3.3 1.3
AP-l-+
TPA 0 0.5 1 2 4 8 12 24 48
(lOng/mi) Time(hour)
1 2.8 2.9 1.9 1.4 1.6 1.9 0.6
AP-1-)
0
.@3
.@
Fig. 4. PDTC inhibits AP-l and NF-.cB transactivation induced by either TPA or
TNF-a. A, PDTC inhibited AP-l transactivation. JB6/AP/KBcells were prepared the same
way as described in Fig 14. For promoterlinhibitor treatment, 3 X l0@cells were cultured
in EMEM with 2% FBS and TPA (10 ng/ml) or TNF-a (10 ng/ml) with or without
inhibitorPDTC(10—40@M).Cellswerethenfurtherculturedfor 18h forTPAtreatment
or 2 h for TNF-a treatment (16-20 and 2—4h were the time periods for maximal
transcriptional activity induction by TPA and TNF-a, respectively, in JB6/AP/icBcells;
Figs. 14 and 3A and data not shown). Luciferase activity was measured as described in
Fig. 2A. AP-l luciferase activity was normalized to the value for the control cells without
promoter/inhibitor treatment. Data are presented as fold induction compared to the
untreatedcontrolcellsasmeansofthreeexperiments;bars,SE.B,PDTCinhibitedNF-KB
transactivation. Cell samples were prepared the same way as described above, and the
NP-KB-CAT activity was normalized to the value of the control cells without promoter/
inhibitor treatment. Data are presented as fold induction compared to the untreated control
cellsas meansof threeexperiments;bars, SE.
Fig.5. SR11302inhibitedTPA-but notTNF
a-induced AP-I and NF-,cB transactivation. A,
SRI 1302 (RA302) inhibited TPA-induced but not
TNF-a-inducedAP-l transactivation.JB6/AP/KB
cells were prepared the same way as described in
Fig 2A. For promoter/inhibitortreatment,cells
wereculturedin EMEMwith 2%FBS andTPA
(10 ng/ml) or TNF-a (10 ng/mI) with or without
inhibitor SR11302 (lO_6 M). Cells were further
cultured for 18 h (for TPA treatment) or 2 h (for
TNF-a treatment). The same samples were used
for assay of both reporters. AP-l luciferase activity
was normalized to the value for the control cells
without promoter/inhibitor treatment. Results are
presented as fold induction compared to the Un
treated control cells as means of three experiments;
bars, SE. B, SR1 1302 (RA302) inhibits TPA-in
duced but not TNF-a-induced NF-.cB transactiva
tion. Cell samples were prepared the same way as
described above, and the NF-,cB-CATactivity was
normalized to the value of the control cells without
promoter/inhibitor treatment. Results are presented
as fold induction compared to the untreated control
cellsas meansof threeexperiments;bars. SE.
induced by TPA was also inhibited by treatment with the retinoid
SR1 1302, paralleling the transactivation inhibition. In contrast,
SRI 1302 did not significantly affect AP-l binding induced by TNF-a
(Fig. 6). These data, together with the transcriptional activation results
(shown in Figs. 4 and 5), support the hypothesis that AP-l transacti
vation by TPA and TNF-a is mediated through different signaling
pathways, with retinoids inhibiting the activation only of TPA-in
duced DNA binding. It is noteworthy that the basal AP-l DNA
binding was not inhibited by either PDTC or SRI 1302 at the indicated
concentrations (data not shown), indicating a selective effect of these
TPA (lOng) . 10 10 10 10 - - . - inhibitors on induced transcription factor binding.
TNF (lOng) . . . . . 10 10 10 10 Different Compositions of AP-1 Complexes Induced by TPA or
PDTC (uM) 0 0 10 20 40 0 10 20 40 . .
TNF-a. Because retlnold transrepression of AP- I occurs after TPA
but not after TNF-a and because this AP-l transrepression is thought
to target c-Jun. we asked whether the c-Jun content might distinguish
the TPA- from the TNF-a-induced complexes. We analyzed the AP-I
components by supershift with the Jun family antibodies using the
nuclear extract purified from JB6/AP/icB reporter cells exposed to
TPA (10 ng/ml for 18 h) or TNF-a (10 ng/ml for 2 h). Fig. 7 shows
the sensitivity to antibody blocking of AP-l binding induced by TPA
and TNF-a. As indicated, Jun D was detected as a major component
consistently present in AP-l complexes in JB6 P+ cells, revealed by
both supershift and blocked DNA binding in the control as well as in
TPA or TNF-a-induced AP-l complexes (Fig. 7 and data not shown).
I
0-s
control
AP-l AND NF-icBIN TRANSFORMATION
A
2
1
o-@
TPA(ng)
TNF(ng)
PDTC(UM)
control
- 10 10 10 10 - - - -
- - - . - 10 10 10 10
0 0 10 20 40 0 10 20 40
following TPA and 2 h following TNF-a. Fig. SA shows that SR1 1302
efficiently inhibited TPA induced AP-l transactivation at the concentra
tions that have previously been shown to inhibit TPA-induced cell trans
formation (23). SR1 1302 also inhibited TPA-induced NF-KB transacti
vation (Fig. SB). Because the retinoids act to prevent AP-l/DNA (Fig. 6)
and c-Jun/DNA (26, 28, 44) binding; this suggests that NF-KB activation
by TPA involves c-Jun or other AP-l protein-dependent signal transduc
tion. In contrast, the same dose of SRI 1302 failed to block TNF-a
induced AP-l or NF-scB transactivation tested from 0 to 48 h following
TNF-a treatment (Fig. 5 and data not shown). This suggests that the
liganded RARs interact with a protein needed for TPA-induced but not
for TNF-a-induced AP-l activation.
PDTC Inhibited AP-1 Binding Induced by Either TPA or
TNF-a, Whereas SR11302 Inhibited Only TPA-induced AP-1
Binding. In parallel with transactivation, the AP-l-DNA binding
induced by TPA (maximal at 8—24h) or by TNF-a (maximal at 1—4
B h)showedsimilartimecourses.DNAbindingofeachtranscription
factor was inhibited by PDTC. Interestingly, the AP- 1 binding activity
B
0
0
.@
0
U
0
a)
0
0
‘S
I-I
IS,
4,
A4
0
..@
0
.@
U
0
to
0
‘S
Gs
D°i
0
4,
0control TPA TPA+RA302 TNF TNF+RA302
Treatment
control TPA TPA+RA302 TNF TNF+RA302
Treatment
3573
AP-l AND NF.KB IN TRANSFORMATION
sitivity of TPA- and TNF-a-induced AP-l transactivation. In contrast,
anti-Jun B produced a blocked shift for TPA but not for TNF-a
induced AP-l complexes (Fig. 7). Increasing the anti-c-Jun antibody
concentration (from 3 to 6 pg/reaction) inhibited more than 50% of
TPA-induced AP-l DNA binding activity, whereas the same concen
tration did not alter the AP-l binding induced by TNF-a. These data
indicate that Jun B and c-Jun. although present at low levels in AP-l
complexes following TPA stimulation, may be important components
responsible for AP-l transactivation by TPA but not by TNF-a. The
presence of c-Jun and Jun B may contribute to the differential sensi
tivity to AP-l transrepression by retinoid shown in Fig. 5.
PDTC Inhibited Transformation by TPA or TNF-a, Whereas
SR11302 Inhibited TPA but not TNF-a-induced Transformation.
To ascertain whether AP-l and/or NF-icB activation are involved in
signaling neoplastic transformation following TPA or TNF-a expo
sure, the anchorage-independent transformation of JB6/AP/scB cells
was determined with or without AP-l or NF-KB inhibitors. Fig. 8A
shows that PDTC efficiently inhibited both TPA- and TNF-a-induced
transformation in a dose-dependent pattern in parallel with the NF-.cB
and AP-l repression shown in Fig. 4. In contrast, the AP-l transre
pressing retinoid SR11302, which efficiently inhibited TPA-induced
AP-l transactivation and cell transformation as reported previously
(23), did not inhibit TNF-a-induced cell transformation (Fig. 8B).
This was in agreement with the lack of transrepression and inhibition
of DNA binding shown in Figs. S and 6. Thus, the differential
sensitivity to retinoid of TPA- and TNF-a-induced JB6 P+ cell
transformation paralleled that seen for AP-l and NF-icB transactiva
tion (Fig. 8). This is compatible with the possibility that activation of
both or either of these transcription factors is required for induction of
neoplastic transformation by either TPA or TNF-a.
DISCUSSION
Both AP-1 and NF-icB, or Either of Them, May Be Involved in
Tumor Promoter-induced Transformation. In the present study
using a JB6 promotion-sensitive AP-l/NF-KB reporter cell line, we
have demonstrated that both AP-l and NF-scB may be involved in
signaling tumor promoter-induced cell transformation, because inhib
itors of either transcription factor blocked transformation. Independ
ent evidence suggesting a requirement for both transcription factors
36 36
— — + +
+ + —
PDTC
SR11302
TNF-ct
— + +
— — — + + —
— — + — + — +
TPA -+ - + - + -
I 3.6 2.8 1.1 3.0 0.8 1.2
AP-1-'
I 2 3 4 5 6 7F
Fig. 6. PDTC inhibited AP-l DNA binding activity induced by either TPA or TNF-a,
whereas RAI 1302 inhibited the AP-l binding induced only by TPA. JB6/AP/icB cells
(5 X 106) were plated into 100-mm cell culture dish exposure to TPA (10 ng/ml) or
TNF-a (10 ng/ml), as described in Fig. 4 and 5, with or without inhibitors SR1I3O2 (lO6
M) or PDTC (40 )LM). Three @zgof nuclear protein isolated from untreated control and
promoter/inhibitor-treated cells as indicated were incubated with 3 X 10―cpm 32P-labeled
collagenase AP-l oligonucleotides and separated by 6% acrylamide gel electrophoresis.
Quantitation of binding activity was the sameas described in Fig. 2 and normalizedto the
value for untreated control cells (top). F. free probe control. Similar results were obtained
using separate nuclear extracts.
The pattern of Jun D supershifting and blocking was identical for
TPA- and TNF-a-induced AP-l complexes, indicating that Jun D,
although a major component in AP-l complexes, is not the rate
limiting signal mediator responsible for the differential retinoid sen
Fig. 7. Different composition of AP-l complexes
induced by TPA or TNF-a in JB6/AP/scB cells. To
determine possible differences in AP-l complexes in
duced by TPA and TNF-a, supershift EMSA of AP-I
with Jun family antibodies specific for c-Jun. JunB, or
JunD were performed with the nuclear extracts from
cellspretreatedwithTPA(10ng/ml,18h)orTNF-a(10
ng/ml, 2 h) as described. Three @gof nuclear protein
isolated from control or promoter/inhibitor-treated cells
were first incubated with antibodies to c-Jun. JunB, or
JunD at indicated concentrations for 20 mm; then
3 X 10―cpm32P-labeledcollagenaseAP-Ioligonucleo
tide was added into each reaction, and EMSA was
performed in 6% acrylamide gel electrophoresis. Ar
rows, supershifts and AP-l DNA bindings; F. free
probe. An additional EMSA experiment using separate
nuclear extracts gave similar results.
c-Jun JunB JunD c-Jun
antibody
(ug)
TNF-a 1 3 1 3 1 3 1 3 0.5 1 0.5 1
— — + — — + + — — + + — — + +
TPA — + — + + — — + + — — + + — —
12 3 4 5 6 7 89 10111213141SF l61718l9F
supershift —°
AP-l-a
3574
. SI...
AP-l AND NF-,cB IN TRANSFORMATION
A
V
@ 1.2
0
@‘ 1.0—
0
a
‘S
0
I
TPA TPA+P1OTPA+P2OTPA+P40 TNF TNF+PSOTNF+P20 TNF+P40
1.2 ———@— - — —---- ____________
0.0 TPA TPA+SR.7 TPA+SR.6 TPA+SR.S TNF TNF+SR.7 TNF+SR.6 TP4F+SR.S
Fsg. 8. Differential inhibition of cell transfo.mation by PDTC or SR1 1302. 1B6/AP/scB
reporter cell line was exposed to TPA (3 ng/ml, 5 nat) or TNF-a (10 ng/ml) with different
concentrations of PDTC (A; P. PDTC; PlO, P20, and P40, 10, 20, and 40 psi, respectively)
or with different concentrations of retinoid SRi 1302 (B; SR. SRI 1302; SR-7, SR-6, and SR-S.
l0@, l06,@ lO@ M,respectively). Cells from each promoterlmhibitor treatment were
suspended in 033% agar (1,000-10,000 cells/dish), and the anchorage-independent colonies
were scored after 14 days incubation at 36°Cwith 5% CO2@ 100% humidity. Data
presented as the mean of three experiments show the relative transformation frequency
compared to the value for TPA or TNF-a treatment only; bars, SE.
has come from our observations that dominant-negative jun expres
sion in mouse (7) or human (8) keratinocytes blocked not only AP-l
but also NF-KB activation when progression was also blocked. Tran
srepression of AP-l and NF-.cB was relatively specific because p53-
dependent transactivation was unaffected by the mutant c-jun expres
sion. This NH2-terminally truncated dominant-negative Jun (TAM67)
with an intact leucine zipper binds specifically to leucine zippers of
endogenous Jun and Fos family proteins, forming low-activity AP-l
complexes (6). TAM67 binding would be expected to render the c-Jun
and the c-Fos bZip domains unavailable to p65, which requires the
bZip domain for interaction (20). Alternatively, TAM67 might inter
act via its bZip domain with p65, rendering the p65 unavailable to
interact with c-Jun or c-Fos. This inhibition by TAM67 of NF-KB
suggests that NF-KB activation in the mouse and human keratinocyte
models requires intact c-Jun and/or c-Fos. Because TAM67 inhibits
both NF-KB and AP-l activation when it blocks neoplastic progres
sion, these experiments suggest but do not prove a role for both
transcription factors in signaling tumor promoter-induced neoplastic
transformation. Because in the current studies small molecule inhib
itors of NF-.cB or AP-l block activation of both or neither transcrip
tion factors, with paralleloutcomesfor transformation,neither Iran
scription factor can be excluded as required for the transformation
response.
NF-icB Activation Occurs Early and May Mediate AP-1 Trans
activation in TPA-induced Transformation. Because NF-KB re
sponds earlier, at least to TPA (compare Fig. 2A and 3A), it may act
as a mediator of later AP-l transactivation, which in turn is essential
for transformation. Cross-interaction of transcription factors including
AP-l and NF-KB has been described (20, 45). Components of AP-l,
c-Jun. or c-Fos, but not Jun B or Jun D, as well as components of
NF-KB, p65 but not p50 or cRel, cross-interact and synergize in both
AP-l and NF-KB-dependent gene transactivation (20). The interaction
involves the bZip domain of c-Jun or c-Fos and the rel homology
domain of p65. The p65/c-Jun or p65/c-Fos interaction-induced trans
activation appears specific for the AP-1PFPA response element be
cause the combination of p65 and Fos, or p65 and Jun. produced little
activation of a reporter driven by a human c-fos promoter containing
a serum-response element (20), and no synergistic effect was detected
between p65 and CREB or Myb (20). Because the antioxidant PDTC
appears to work by preventing 1KB release from NF-KB proteins,
resulting in blocked nuclear entry, the observation that AP-l is also
inhibited suggests a dependency of AP-l activation on interaction
with NF-scB p65 in the nucleus. In the JB6 model, this effect would
appear to predominate over that described by Schenk et al. (33), who
found that in HeLa and L929 cells, antioxidants such as PDTC
actually activate AP-l DNA binding and transactivation. The latter
effect required new synthesis of c-Fos and c-Jun proteins. The fact
that an inhibitor of NF-KB such as PDTC also inhibits AP-I and
transformation in the JB6 model suggests dependency of AP-l acti
vation on NF-KB activation and dependency of transformation on
AP-l activation. However, because PDTC may act far upstream of
1KBrelease, we cannot exclude the possibility that PDTC independ
ently inhibits AP-l transactivation in the JB6 model. To clarify the
role and consequences of NF-KB inhibition, the use of more specific
NF-KB inhibitors, such as 1KB or antisense p65, should be informa
tive.
The AP-1 Complex Induced by TNF-a Differs from That In
duced by the Phorbol Ester TPA. AP-l activity is down-regulated
by a group of liganded nuclear receptor family proteins including
RAR andglucocorticoidreceptor(27, 44, 46, 47). The presentresults
show, for the first time to our knowledge, the inhibitory effect of the
synthetic anti-AP-l retinoids on TPA-induced AP-l DNA binding and
transactivation (Figs. 5, 6, and 8). The retinoid did not, however,
inhibitTNF-a-inducedAP-l activity.RA-ligandedRARa, (3,ory,
but not retinoid X receptor, has been shown to block AP-l DNA
binding and transactivation (26, 28). Unlike the case for the interac
tion between glucocorticoid receptor and AP-l (27), RAR/c-Jun in
teraction domains have not been determined, and evidence for direct
RAR/c-Jun interaction has not emerged. The insensitivity to retinoid
of TNF-a-induced but not TPA-induced AP-l activation suggested
different compositions of the AP- 1complexes. We have demonstrated
that the TPA-induced but not the TNF-a-induced AP-l complex
contained Jun B and c-Jun in addition to Jun D, which was found in
both cases (Fig. 7). The lack of these components, particularly c-Jun.
may explain the resistance to transrepression of AP-l by RA follow
ing TNF-a exposure because RA inhibited the binding of c-Jun
homodimers to DNA (28). This lack of c-Jun in the AP-l complex
induced by TNF-a would not be expected to exclude the possible
cross-interaction between NF-,cB and AP- 1 subunits because p65/c-
Fos interaction still could occur. The presence of c-Jun in TPA
induced AP-l complexes may explain retinoid sensitivity and facili
tate the cross-interaction with NF-icB p65, which fails to interact with
Jun D or Jun B. Thus, c-Jun may be a limiting factor when AP-l
dependent signal transduction and transformation is inhibited by ret
inoids.c-Junmay,however,notbetheonlycriticalfactorcontributing
to the regulation of AP-l activation and its transrepression by reti
noids. Investigating the possible limiting role of the recently described
CREB-binding protein (48—50)as well as that of c-Jun may prove
3575
1I@
AP-I AND NF-.cB IN TRANSFORMATION
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enlightening for understanding the differential sensitivity to retinoid
transrepression.
In conclusion, the present data from a JB6 transformation-sensitive
cell line show that both AP-l and NF-,cB may be involved in signaling
tumor promoter-induced transformation. The data do not exclude
either cross-interacting or independent pathways. The signal pathway
initiated by TNF-cx differs from that initiated by the phorbol ester TPA
by an apparent bypass of c-Jun and Jun B in the TNF-a-induced AP-l
complex.
ACKNOWLEDGMENTS
We acknowledge Dr. Marcia I. Dawson for providing the SR11302 RA,
Drs. Antonio Sica and Howard Young for providing the IL-6 NF-KB-CAT
reporter plasmid, and Dr. Nancy Rice for insightful and critical reading of the
manuscript.
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