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INTRODUCTION |
Programmed cell death (apoptosis) is a common cellular response to
stress caused by environmental challenges (1). Altered expression of
apoptosis-related proteins, which coincides with decreased or absent
apoptosis, is commonly observed in various tumor types and is of
fundamental importance in tumor resistance to host defenses as well as
to clinical therapy (2). Indeed, reduced ability of tumors to undergo
apoptosis is often associated with elevated drug resistance and poor
clinical outcomes. Malignant melanoma is a primary example of a cancer
that responds poorly to various treatments, including chemotherapy and
-irradiation (3). Despite the alarming increase in the incidence of
this tumor in the past decade, the molecular mechanisms of its
progression as well as the regulation of apoptosis in human melanoma
remain largely unknown (4).
The mechanisms underlying apoptotic signaling have been intensively
studied in recent years. Two cell surface molecules, Fas and
TNFR1,1 represent the main
death signaling receptors (1). Receptor trimerization by ligands FasL
and TNF
, respectively, initiate receptor interaction with the
intermediate signaling molecules FADD and FLICE (caspase-8), followed
by activation of the caspase cascade (1, 5, 6). In addition to its role
in apoptosis, TNFR1-mediated signaling is also linked with the
regulation of survival functions, including induction of cytokines,
growth factors, and cell proliferation and differentiation (7-9).
Furthermore, TNF
appears to be one of the primary growth regulators
of metastatic cancer cells (10, 11), including late-stage melanoma
cells (12-14). Hence, suppression of TNF signaling may enhance the
apoptotic stimulus to tumor cells. Key to understanding the interplay
between TNF
and Fas signaling is the nature of their upstream
transcriptional regulators and downstream targets (i.e.
inhibitors of apoptosis and survival proteins) in concordance with, or
independent of, NF-
B-dependent signaling (15-17).
Expression of the human TNF
gene is controlled by the AP-1, ATF2,
Erg-1, C/EBP
, and NF-
B transcription factors (18-22).
Transcriptional regulation of Fas and FasL genes was shown to be
mediated by NF-
B, AP-1, NF-AT, ATF2, and Egr3 (23-26). As a dynamic
process, programmed cell death is dependent, in many cases, on new gene
expression (27), and is tightly regulated at the transcriptional level
(28-30).
Our interest in exploring mechanisms underlying key melanoma phenotypes
led us to identify CREB/ATF transcription factors as regulatory
proteins that play important roles in determining both melanoma's
resistance to radiation and its metastatic potential (31, 32). ATF2, a
member of the ATF/CREB family of basic region leucine zipper (bZIP)
DNA-binding proteins (33-35), was shown to modulate melanoma's
resistance to UVC irradiation but not to contribute to its metastatic
potential (36). ATF2 is among several transcription factors, including
AP-1, CREB, and Rel/NF-
B, which have been found to be UV-inducible.
ATF2 induction in response to stress occurs at the levels of
transcription-translation and posttranslational modification, the
latter of which is mediated by JNK/p38 kinases (37, 38). ATF2 has been
implicated in the transcriptional control of various stress-responsive
genes, including c-Jun (39), interferon-
(40), urokinase (41),
TGF-
2 (42), TNF
(18, 20), and DNA polymerase
(43).
Differential splicing of ATF2 creates several isoforms which exhibit
different transcriptional outputs (44). Full-length ATF2 is
transcriptionally inactive in its native form as a result of
intramolecular interaction of its DNA-binding domain with the
amino-terminal transactivation domain (33). ATF2 as well as a related
factor, ATFa, not only can form DNA-binding homodimers but also can
efficiently heterodimerize with members of the ATF/CREB and Jun/Fos
families, thus providing an important transcriptional control mechanism
(41, 45-47). Duration and magnitude of ATF2 transcriptional output are
also regulated at the level of phosphorylation and stability. While
stable in its native non-active form, ATF2 is a short-lived protein
after homo- or heterodimerization. ATF2 instability is further
facilitated by heterodimerization with certain partners, which provide
additional targeting molecules for ubiquitination through their own
docking sites, as shown for c-Jun (48).
To identify mechanisms underlying ATF2's ability to modulate radiation
resistance, we studied the possible role of ATF2 in apoptosis of human
melanoma cells. We demonstrate that ATF2 expression increases
UV-mediated apoptosis through suppression of TNF
expression, which
elicits survival signals in tumors of this type.
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EXPERIMENTAL PROCEDURES |
Cell Lines--
Human melanoma LU1205 cells were maintained in
MCDB153/L15 medium (4:1) supplemented with 5% fetal bovine serum,
L-glutamine, and antibiotics. LU1205/neo, LU1205/ATF2,
LU1205/ATF2
1-195, and LU1205/I
B
cell lines were maintained in
the same medium supplemented with G418 (200 µg/ml). Cells were grown
at 37 °C with 5% CO2.
Stable Transfection and Selection--
The expression vectors
pECE-ATF2 encoding full-size ATF2 and pECE-ATF2
1-195, a truncated
form of ATF2 cDNA lacking the first 195 amino acids and fused with
GAL4 (34, 35), were transfected by electroporation (230 V, 1050 microfarads) into LU1205 cells together with pBK-RSV-neo plasmid
(Stratagene, La Jolla, CA) as described previously (36). In parallel,
the pECE vector was cotransfected with pBK-RSV-neo to generate control
LU1205/neo cells. The expression vector pCMV4-I
B
(49) plus
pcDNA3-neo (Invitrogen, Carlsbad, CA) was also cotransfected by
electroporation into LU1205 cells. LU1205/neo, LU1205/ATF2,
LU1205/ATF2
1-195, and LU1205/I
B
cell lines were created as a
mixed population of G418-resistant clones.
Transient Transfection and Luciferase Assay--
Reporter
constructs (20 µg) were transiently cotransfected with the indicated
expression vectors and pCMV-
-gal (5 µg) into 107
LU1205 melanoma cells by electroporation (230 V, 1050 microfarads; Gene
Pulser, IBI). The reporter constructs used were: 5xJun2tk-Luc and
vector tk-Luc (50);
615 TNF-Luc,
615 TNF(mutCRE)-Luc,
615
TNF(mutAP-1)-Luc,
36 TNF-Luc (19),
453 FasL-Luc,
318 FasL-Luc,
237 FasL-Luc (23),
1.3kb FasL-Luc, and variants of this construct
with mutations in the NF-
B- and AP-1 sites (24). Expression
constructs pECE-ATF2, pECE-ATF2
1-195 (34, 35), pCMV-c-Jun-HA (51),
and TAM67, a dominant negative form of c-Jun (52, 53), were also used
in these experiments. Luciferase activity was determined using the
Promega luciferase assay system (Promega, Madison, WI). Luciferase
activities were normalized on the basis of
-galactosidase levels in
transfected cells.
Transient Transfection and GFP Assay--
Melanoma cells (6 × 106) were transiently cotransfected with either ATF-2-
or TAM-67 expression vectors together with marker plasmid encoding
green fluorescent protein, pGFP (CLONTECH), 10 and
2.5 µg, respectively, by electroporation. Twenty-four hours after
transfection cells were irradiated by UVC (60 J/m2) and
18 h after treatment were stained with PI and analyzed using flow
cytometry. The ratio of GFP+PI+ to total
GFP+ cells was used to measure frequencies of death in the
transfected cells.
Transient Transfection and X-gal Staining--
Melanoma cells
(6 × 106) were transiently cotransfected with ATF2
expression vectors and pCMV-
-gal (10 and 2.5 µg, respectively) by
electroporation. Twenty-four hours after transfection, cells were
irradiated with UVC (60 J/m2) as described previously (32).
To identify
-galactosidase activity, cells were fixed 18 h
after UVC treatment and stained with X-gal (28). The ratio of blue
cells with apoptotic morphology to the total number of blue cells
served as the indicator of the level of apoptosis in transfected cells.
Cell Treatment and Apoptosis Studies of Stably Transfected
Melanoma Cells--
Cycloheximide (10 µg/ml), actinomycin D (200 ng/ml) (Sigma), and the caspase inhibitor, zVAD-fmk (10-50
µM) (Enzyme Systems, Dublin, CA) were added as indicated.
The proteasome inhibitor lactacystin was purchased (Dr. E. J. Corey, Harvard University, MA) and used at 10 µM.
Recombinant TNF
(PharMingen, San Diego, CA) was used at a final
concentration of 1-4 ng/ml. Antagonistic monospecific antibodies
against Fas (clone G254-274 from PharMingen) and against TNFR1 (clone
16803.1 from R&D Systems, Minneapolis, MN) were added as indicated at
the final concentration of 1-5 µg/ml.
Annexin-V-FITC staining in the presence of PI was performed for
detection of early apoptosis levels using TACS Annexin-V-FITC kit
(Trevigen, Gaithersburg, MD). Flow cytometric analysis was performed on
an Epics-Profile II flow cytometer (Coulter, Hialeah, FL) with Elite
software 4.01 by analyzing 104 cells/sample, using wide
scatter gates to include late apoptotic cells.
DNA fragmentation analysis was performed as described previously (54).
Cells were pelleted and resuspended in 0.5 ml of hypotonic buffer with
0.1% Triton X-100 containing propidium iodide (PI) (40 µg/ml) and
DNase-free RNase A (1 mg/ml). Cells were incubated at 37 °C for 30 min and analyzed on an Epics-Profile II flow cytometer (Coulter). The
percentage of cells to the left of the diploid G0/1 peak,
diagnostic of hypodiploid cells that have lost DNA, was taken as the
percentage of apoptotic cells. The analysis was performed without light
scatter gating.
Western Blotting Analysis--
Total cell extracts were resolved
on 10% SDS-polyacrylamide gel electrophoresis, transferred to
nitrocellulose, and processed by standard methods. The polyclonal
anti-PARP serum (Biomol, Plymouth Meeting, PA) was used at a 1:1000
dilution. Polyclonal Abs against ATF2 and phosphoATF2 (New England
Biolabs) were used at a 1:1000 dilution. Monoclonal Abs against human
TNF
(clone Mab1), Fas (clone G254-267) (PharMingen), and TNFR1(R&D
System), and polyclonal Abs against FasL and I
B
(Santa-Cruz
Biotechnology) were used at the same dilution. The secondary Abs were
goat anti-rabbit IgG or goat anti-mouse IgG conjugated to horseradish
peroxidase (dilution 1:5000). Signals were detected using the ECL
system (Amersham Pharmacia Biotech).
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RESULTS |
Characterization of UVC-induced Apoptosis in Human Melanoma
Cells--
To study regulation of UVC-induced apoptosis in melanoma,
we used the LU1205 cell line, which represents a late stage melanoma that harbors wild type p53. These cells exhibit higher resistance to
irradiation than early stage melanoma cells (55). The finding that
expression of a truncated ATF2 form (
1-195) reduced radiation resistance of late-stage melanoma cells (36) prompted our interest in
elucidating mechanisms by which ATF2 may elicit such changes. Since
radiation resistance may be inversely correlated with the degree of
programmed cell death, we studied the possible role of ATF2 in
UV-induced apoptosis of these melanoma cells.
Several markers of apoptosis were used to monitor and characterize
UVC-mediated programmed cell death in melanoma cells. To distinguish
between early and late phases of apoptosis, annexin-V-FITC and PI
staining, which detect early and late phases of apoptosis (56),
respectively, were used. Eighteen hours after UVC irradiation, 20% of
LU1205 melanoma cells were annexin-V+, of which 11% were
annexin-V+PI
and 9%
annexin-V+PI+, reflecting the early and late
phases of apoptosis, respectively (Fig.
1A). In several independent
experiments, we found that 25 ± 6% LU1205 cells were
annexin-V+ (apoptotic) 18 h after UVC irradiation,
compared with 5 ± 3% for nontreated cells. DNA fragmentation
analysis, performed by staining cell nuclei with PI and determining the
percentage of apoptotic cells with hypodiploid DNA content (Fig.
1B), revealed that 36% of the total cell population had
apoptotic nuclei with hypodiploid DNA content 40 h after UVC
irradiation, compared with 2% for nontreated cells (Fig.
1B). Within 3 days after treatment the apoptosis level
reached 70%; the vast majority of melanoma cells died 4 days after
irradiation (data not shown). Cycloheximide (Fig. 1C) as
well as actinomycin D (data not shown) partially suppressed UVC-induced
apoptosis of melanoma cells, indicating that UVC-induced gene
expression is required if maximal levels of apoptosis are to be
achieved.

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Fig. 1.
UVC irradiation induces melanoma cell
apoptosis. A, LU1205 cells were treated with UVC at 60 J/m2 in the presence of zVAD-fmk (50 µM) and
lactacystin (10 µM), added 1 h before irradiation
and also in the absence of these materials. The percentage of early
apoptosis was determined 18 h after UVC treatment using
annexin-V-FITC plus PI staining and flow cytometry. The percentage of
stained cells is shown near each quadrant. Quadrant
3 contains Annexin-V PI (live)
cells. Annexin-V+PI and
annexin-V+PI+ cells indicate early and late
phases of apoptosis, respectively. B, DNA fragmentation
analysis of LU1205 cells was performed 40 h after UVC treatment,
as described under "Experimental Procedures." The percentage of
cells in gate 1 indicates the level of apoptotic cells with hypodiploid
DNA content. C, effect of cycloheximide (CHX) (10 µg/ml)
on UVC-induced apoptosis of LU1205 cells. The percentage of early
apoptosis (annexin-V+PI ) is shown.
D, UVC-induced cleavage of PARP. Western blot analysis of
PARP was performed 18 h after UVC irradiation (40 J/m2
for lane 1 and 60 J/m2 for
lanes 2-7) of LU1205 cells in absence or
presence of zVAD-fmk (50 µM), lactacystin (10 µM) or PMA (10 ng/ml). The position of intact (116 kDa)
and cleaved (85 kDa) forms of PARP is indicated by
arrows.
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Since caspase-dependent proteolysis has been shown to play
an important role in different types of programmed cell death (5), we
determined the possible role of activation of the caspase cascade following UVC irradiation of melanoma cells. A 50 µM
quantity of zVAD-fmk, a caspase inhibitor, substantially suppressed
UVC-induced apoptosis (Fig. 1, A and B). To
monitor cleavage of poly(ADP-ribose) polymerase (PARP), a typical
target of caspase-3 and an indicator of early apoptotic commitment
(57), immunoblot analysis of nuclear proteins from UVC-treated melanoma
cells was performed with the aid of anti-PARP Ab, revealing the
presence of the 85-kDa cleavage product 18 h after UVC treatment,
but not in the presence of zVAD-fmk (Fig. 1D).
The role of proteasome-dependent degradation in
UVC-mediated apoptosis of melanoma cells was examined using the
proteasome inhibitor lactacystin (58). Whereas 10 µM
lactacystin did not induce apoptosis in nonstressed melanoma cells
(data not shown), a substantial increase in UVC-induced apoptosis was
seen in the presence of 10 µM lactacystin, indicating a
protective role of proteasome-dependent protein degradation
in UVC-induced apoptosis (Fig. 1A). Increased PARP cleavage
was seen in lactacystin-treated cells following UVC irradiation (Fig.
1D). These observations suggest that all three major
cell regulatory processes (new gene expression,
proteasome-dependent protein degradation, and caspase proteolysis) contribute to UVC-induced apoptosis of melanoma cells.
Role of ATF2 in UVC-induced Gene Response and Apoptosis of Human
Melanoma Cells--
To elucidate a probable function of ATF2 in the
regulation of UVC-induced apoptosis of human melanoma cells, we
utilized expression vectors encoding either GAL4 fused with truncated
ATF2 lacking the transactivation domain (pECE-ATF2
1-195) or
full-size ATF2 (pECE-ATF2) (34, 35). To monitor ATF2-mediated
transactivation, a reporter construct containing five repeats of the
ATF-2/AP-1 binding site from the Jun promoter (TRE-Jun2), which
preferentially bind ATF2/Jun heterodimers (39, 50), was used.
ATF2/AP-1-dependent luciferase activity increased 3-fold
after UVC irradiation of control LU1205 cells. Transient transfection
of truncated ATF2 (at a 2:1 ratio of expression vector to reporter
construct) slightly increased the basal level of
ATF2/AP-1-dependent transcription but decreased UVC-induced
transactivation of ATF2/AP-1 (25-30%, compared with control cells;
Fig. 2A). In contrast,
expression of full-size ATF2 up-regulated the basal level of
ATF2/AP-1-dependent luciferase activity 2.5-fold.
Unexpectedly, the transfected full-size ATF2 also decreased UVC-induced
ATF2/AP-1-mediated transactivation (Fig. 2A). These
observations suggest that both truncated and full-size ATF2 after
transfection exert a certain silencing effect on UVC-induced
ATF2/AP-1-dependent transcription in melanoma cells.

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Fig. 2.
Modulation of ATF2-dependent
transcription affects UVC-induced apoptosis of melanoma cells.
A, 8 × 106 LU1205 cells were transiently
cotransfected with the reporter construct 5xJun2tk-Luc (5 µg) and
expression vector pECE, pECE-ATF2, or pECE-ATF2 1-195 (10 µg) in
the presence of pCMV- -gal (5 µg). Cotransfection with tk-Luc (5 µg) was used as a control for basal luciferase activity. Transfected
cells were divided into two plates. Twenty-four hours after
transfection, half the plates were irradiated with UVC (60 J/m2). After an additional 18 h, irradiated and
nontreated cells were analyzed for luciferase and -galactosidase
activities. The normalized ratio of luciferase activity to
-galactosidase is shown. B, LU1205 cells were transiently
transfected with pECE, pECE-ATF2, or pECE-ATF2 1-195 (20 µg) in
the presence of pGFP (5 µg). Transfected cells were divided into two
plates. Twenty-four hours after transfection, half the plates were
irradiated with UVC (60 J/m2). After an additional 18 h, irradiated and nontreated cells were stained with PI and analyzed by
flow cytometry. The ratio of PI+ GFP+ cells to
all GFP+ cells indicates the frequency of apoptotic cells
in the population of transfected cells.
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We next determined whether transcriptional suppression by ATF2 alters
the degree to which melanoma cells undergo apoptosis in response to UV
irradiation. Apoptosis studies were performed by cotransfection of ATF2
expression vectors with either pGFP or pCMV-
-gal encoding green
fluorescent protein and
-galactosidase, respectively, as marker
genes. To determine the frequency of death in ATF2-transfected cells,
UVC-irradiated and nontreated cells were stained with PI and analyzed
by flow cytometry, allowing quantification of
GFP+PI+ cells in the overall population of
GFP+ cells. Expression of either full-length or truncated
forms of ATF2 notably increased (1.8- and 1.3-fold, respectively) the
frequency of cell death following UVC irradiation of LU1205 cells (Fig. 2B). Parallel analysis using X-gal staining of
-galactosidase-expressing cells enabled visual detection of blue
cells with apoptotic morphology in the total population of blue cells
(28), revealing a similar increase in apoptosis levels after UVC
irradiation in ATF2-transfected cells (data not shown).
To further investigate ATF2-dependent regulation of
UVC-induced apoptosis of human melanoma cells, we established LU1205
cell lines stably transfected with either full-size ATF2 cDNA
(LU1205/ATF2 cells) or the truncated form, GAL4-ATF2
1-195
(LU1205/ATF2
1-195), as well as the control cell line transfected
with the empty vector and neo marker plasmid (LU1205/neo). Western blot
analysis confirmed elevated expression of the respective forms of ATF2.
Cells that constitutively express the full-size ATF2 revealed increased
levels of 68-kDa ATF2 as well as spliced isoforms, before and after UVC irradiation (Fig. 3A).
However, immunoblot with anti-phospho-ATF2 (Thr71) Ab revealed the same
level of phosphorylated forms of ATF2 after UVC irradiation of
LU1205/ATF2 cells, indicating a relative decrease in the ratio of
phospho-ATF2 to total ATF2 (Fig. 3A). Immunoprecipitation
with anti-GAL4 and subsequent Western blotting with anti-ATF2 Ab
further confirmed the presence of the fused truncated form of ATF2 in
LU1205/ATF2
1-195 cells (data not shown), as was observed previously
with MeWo melanoma cells transfected with ATF2
1-195 (36).

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Fig. 3.
A, total extracts of stably transfected
cell lines LU1205/ATF2 and LU1205/neo were analyzed by Western blotting
with anti-ATF2 and anti-phospho-ATF2 Abs. Positions of p68, p55, p50,
and p42 forms of ATF2 and phosphorylated p68* p50*, and p42* forms are
indicated by arrows. B, effect of modulation of
ATF2 levels in stably transfected melanoma cell lines (LU1205/ATF2,
LU1205/ATF2 1-195, and LU1205/neo) on transcriptional activity of
the ATF2-dependent reporter constructs. 107
LU1205/neo, LU1205/ATF2, and LU1205/ATF2 1-195 cells were
transiently transfected with 5xJun2tk-Luc (20 µg) in the presence of
pCMV- -gal (5 µg). Twenty-four hours after transfection, cells were
irradiated with UVC, and, after an additional 18 h, luciferase and
-galactosidase activity were determined. The normalized ratio of
luciferase to -galactosidase activity is indicated.
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We next examined transcriptional regulation of a reporter construct
containing ATF2/AP-1 binding sites (5xJun2tk-Luc) in LU1205/ATF2 and
LU1205/ATF2
1-195 cells. In agreement with the results of transient
transfection studies, LU1205/ATF2 cells possessed increased basal
levels of ATF2/AP-1-dependent luciferase activity and
decreased UVC-induced levels of this activity (30-35%) compared with
control cells. UVC-induced ATF2-dependent luciferase
activity was also decreased (20-25%) in LU1205/ATF2
1-195 cells
(Fig. 3B).
To monitor apoptosis in cell lines stably transfected with ATF2
constructs, we used Annexin-V staining and DNA fragmentation analysis.
As found in the apoptosis study of transiently transfected cells,
annexin-V plus PI staining (Fig.
4A) showed increased levels of
cell death in both LU1205/ATF2 and LU1205/ATF2
1-195 cells compared
with the control LU1205/neo cells 16-24 h after UVC irradiation. UVC-induced apoptosis levels (annexin-V+) were enriched
21 ± 2%, 30 ± 3%, and 32 ± 3% relative to control LU1205/neo, LU1205/ATF2
1-195, and LU1205/ATF2 cells, respectively, 24 h after UVC irradiation (60 J/m2), based on the
results of four independent experiments. DNA fragmentation analysis
showed an additional increase of apoptosis levels 40 h after UVC
treatment for LU1205/ATF2 (64 ± 10%), compared with LU1205/ATF2
1-195 (31%) and control (24 ± 4%) cells (Fig.
4B). These observations suggest that expression of either
truncated or full-size ATF2 increase the level of UV-induced apoptosis
in LU1205 melanoma cells. Interestingly, full-length ATF2 elicited a
greater increase than the amino-terminal truncated form.

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Fig. 4.
UVC-induced apoptosis of LU1205 cells stably
transfected with ATF2 constructs. LU1205/neo, LU1205/ATF2, and
LU1205/ATF2 1-195 cells were irradiated with UVC (60 J/m2). A, apoptosis levels were determined using
annexin-V-FITC plus PI staining 3-24 h after treatment. B,
DNA fragmentation analysis was performed 40 h after treatment as
described under "Experimental Procedures."
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Effect of ATF2 on UVC-induced TNF
Expression--
Metastatic
melanomas actively produce a set of cytokines, including TNF
, that
appear to provide autocrine growth conditions (13, 14, 59). Indeed,
luciferase activity driven by TNF
promoter (
615 TNF-Luc) (19), was
up-regulated (5-7-fold) after UVC irradiation of LU1205 cells (Fig.
5A). Mutation in the CREB/AP-1 site (at the
106 position) of this construct partially abrogated this
increase (by 40-60%). In contrast, mutation within the AP-1 binding
site (at the
66 position) was less pronounced (Fig. 5A), suggesting that the CREB/AP-1 site (at
106) is the primary regulator of TNF
expression in melanoma cells, possibly via ATF2, as was described previously for other cell systems (18-20).

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Fig. 5.
A, effect of mutations of the CRE
( 106) and AP-1 ( 66) sites on TNF promoter activity in LU1205
cells. LU1205 cells were transiently cotransfected with 615 TNF-Luc,
615 TNF(mutCRE)-Luc, or 615 TNF(mutAP-1)-Luc reporter constructs.
B, effect of altered ATF2 levels on TNF promoter
activity. 107 LU1205 cells were transiently cotransfected
with 615 TNF-Luc reporter construct (5 µg) and with indicated
amounts of the expression vectors pECE-ATF2, pECE-ATF2 1-195, or
with pCMV-TAM67, a dominant negative mutant of c-Jun, in the presence
of pCMV- -gal (5 µg). The total quantity of DNA for transfection
was adjusted to 30 µg with vector DNA. The normalized ratio of
luciferase to -galactosidase activity was determined 18 h after
UVC treatment.
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To confirm the role of ATF2 in regulation of TNF
expression in the
melanoma cells, LU1205 cells were transiently cotransfected with the
615 TNF-Luc reporter construct and either full-size or truncated ATF2
expression vectors. Both ATF2 constructs reduced the degree of
UVC-mediated transactivation by the TNF
promoter in a
dose-dependent manner (Fig. 5B). That
full-length (although hypophosphorylated upon UV irradiation; Fig.
3A) and truncated (without transactivator domain) forms of
ATF2 elicit the same silencing effect on TNF
transcription could be
attributed to squelching of a positive factor as a result of efficient
heterodimerization with other bZIP family members. Among the primary
transcription factors that associate with ATF2 is c-Jun (39, 47), which was also shown to increase TNF
transcription via the CRE/AP-1 site,
following stimulation of monocytes by lipopolysaccharide (21). We
therefore tested the possible effect of c-Jun on TNF
transcription
in ATF2-expressing melanoma cells. To this end transcriptional activity
of c-Jun was blocked by using a dominant negative c-Jun construct,
TAM-67, which lacks the first 200 amino acids (52, 53), similar to the
truncated ATF2 form used in the present study. Expression of TAM67 in
LU1205 suppressed TNF promoter-dependent luciferase
activity (Fig. 5B) as well as increased apoptosis (data not
shown). Conversely, transfection of wild type c-Jun resulted in a
dose-dependent increase of TNF
promoter activity in both non-treated and UVC-irradiated cells (Fig.
6A). Furthermore, forced expression of wild type c-Jun partially abrogated the negative effect
of both full-length and truncated forms of ATF2 on TNF
promoter
activity (Fig. 6A). c-Jun expression in LU1205 cells (cotransfected with the GFP marker plasmid) also blocked the increase in UVC-induced apoptosis caused either by ATF2
1-195 (Fig.
6B) or full-length ATF2 overexpression (data not shown).
These observations suggest that the ability of ATF2 to increase the
degree of UVC-induced apoptosis is mediated via suppression of positive
transcription factors, such as c-Jun.

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Fig. 6.
A, c-Jun transactivates TNF promoter
activity in LU1205 cells. Effect of full-length and truncated forms of
ATF2 on c-Jun-dependent transactivation of TNF promoter
activity. LU1205 cells were transiently cotransfected with 615
TNF-Luc (5 µg) in the presence or absence of 10 µg of pECE-ATF2 and
pECE-ATF2 1-195 and indicated quantities of the pCMV-c-Jun
expression construct and 5 µg of -galactosidase. Luciferase and
-galactosidase activities were determined after UVC treatment, as
described in the legend to Fig. 2. B, c-Jun partially
suppressed UVC-induced apoptosis, which was accelerated by
ATF2 1-195 expression. LU1205 cells were cotransfected with either
pECE-ATF2 1-195 (20 µg) or with this vector and pCMV-c-Jun-HA (10 µg or 20 µg) in the presence of GFP marker plasmid. Two days after
transfection, cells were UVC irradiated. Apoptosis levels of
GFP-positive cells were detected using flow cytometry after PI
staining.
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Analysis of TNF
promoter activity in stably transfected LU1205/ATF2
cells further confirmed the data obtained by transient transfection.
LU1205/ATF2 cells exhibited reduced (35-40%) TNF
promoter activity
after UVC treatment (without changes in basal levels) (Fig.
7A). Decrease in TNF
transcription coincided with down-regulation of TNF
protein levels.
Western blot analysis revealed a UVC-dependent increase in
the level of TNF
protein (p17) in LU1205/neo cells, whereas this
increase was substantially suppressed in LU1205/ATF2 cells (Fig.
7B). Expression of TNFR1 (whose transcription is positively
regulated by TNF
) was also decreased in LU1205/ATF2 cells prior to
and after UV irradiation compared with control cells while TRAF2 (60)
levels were relatively stable (data not shown).

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Fig. 7.
A, determination of TNF promoter
activity in stably transfected melanoma cell lines (LU1205/ATF2,
LU1205/ATF2 1-195, and LU1205/neo). These cells were additionally
transiently cotransfected with 615 TNF-Luc or 36 TNF-Luc constructs
(25 µg). UVC treatment and determination of luciferase activity were
performed as described above. B, Western blot analysis
of TNF and TNFR1/p60 expression in ATF2-transfected LU1205
cells.
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Role of TNFR and Fas-mediated Signaling in UVC-induced Apoptosis of
ATF2-expressing Melanoma Cells--
To determine which death-signaling
pathway mediates UVC-induced apoptosis in control and ATF2-expressing
melanoma cells, antagonistic mAbs to either TNFR1 or Fas that block the
relevant signaling cascade were added to cell cultures 1 h before
UVC irradiation. Addition of anti-TNFR1 mAb further increased the
degree of apoptosis after UVC irradiation of control and ATF2 (both
full-length and truncated forms) transfected cells. Conversely,
anti-Fas mAb substantially suppressed apoptosis in these melanoma cells
(Fig. 8A). These results point
to the pro-apoptotic role of Fas signaling and anti-apoptotic role of
TNFR-mediated signaling for LU1205/neo, LU1205/ATF2, and LU1205/ATF2
1-195 cells following UVC irradiation. To confirm the
protective role of TNFR1-mediated signaling, melanoma cells were
pretreated with recombinant TNF
before UVC irradiation. The presence
of TNF
(2-4 ng/ml) in the medium partially reduced the degree of
UVC-mediated apoptosis of control and ATF2-transfected melanoma cells
(Fig. 8B). These observations demonstrate that TNF
activates a survival signal in melanoma cells, which is suppressed upon
ATF2 expression.

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Fig. 8.
A, effect of antagonistic anti-Fas and
anti-TNFR1 mAbs on UVC-induced apoptosis of control LU1205/neo,
LU1205/ATF2, and LU1205/ATF2 1-195 cells. Cells were pretreated with
5 µg/ml of the indicated mAb 1 h before UVC irradiation (60 J/m2). Abs were maintained in the medium after UVC
treatment. Cells were stained with annexin-V-FITC plus PI and analyzed
using flow cytometry 18 h after UVC treatment. The percentage of
annexin-V-positive (apoptotic) cells is shown. B, effect of
TNF on UVC-induced apoptosis of LU1205 cell lines. Cells were
pretreated with TNF (4 ng/ml) 1.5 h before UVC irradiation.
TNF was maintained in the medium after irradiation. The percentage
of annexin-V-positive (apoptotic) cells is shown.
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Suppression of UVC-induced TNF
promoter activity by both the
truncated and the full-length forms of ATF2, in transient transfection as well as in LU1205/ATF2 cells (followed by down-regulation of TNF
expression), coincided with increased levels of UV-mediated cell death
and pointed to a causal linkage between ATF2 and regulation of cell
death in melanoma. It is important to note, however, that full-length
ATF2 was more efficient in elevating the degree of UVC-induced
apoptosis of melanoma cells than the truncated form of ATF2. Since the
full-length form of ATF2 is also expected to bring about positive
transcriptional output, we performed experiments to determine whether
the apoptotic signal in melanoma cells, namely the Fas pathway, may be
also affected by ATF2. Melanoma cell lines, including LU1205, express
both the death receptor and its ligand, Fas and FasL (Ref. 61 and Fig.
9D). The human FasL promoter contains two NF-AT sites at positions
279 and
140 (23, 62), MEKK-RE
(at
336), which binds ATF2-Jun heterodimers (63), and AP-1- and
NF-
B-binding sites at positions
1088 and
1050, respectively (23,
24). Transfection of nested deletions of 5' FasL promoter constructs
(
1.3 kilobase pairs, and
453,
318,
237 base pairs) revealed
activation of a UVC-dependent promoter. Increased FasL promoter activity after UV irradiation is likely to be mediated by
target sequences located within the
453 and
318 region, as the
237 construct no longer exhibited strong activation by UV irradiation. Further support for the role in FasL activation of the
MEKK-RE site located within this comes from analysis of 5' promoter
sequences mutated within the NF-
B or AP1 sites; neither was able to
completely abrogate UV responsiveness (Fig. 9B). These results point to the role of MEKK-RL and its associated proteins in
mediating increased FasL transcription. Indeed, cells expressing the
ATF2 construct exhibited a higher basal level of FasL promoter activity. After UV irradiation, a further increase of 2-3-fold was
noted in the LU1205ATF2 cells (Fig. 9C). The overall level of FasL protein was also higher in LU1205/ATF2 cells before and after
UVC treatment than in control cells (Fig. 9D). These
observations suggest that ATF2 plays a positive role in the regulation
of FasL transactivation/expression in melanoma (Fig. 9,
A-D), as previously observed in other cell systems (63).
Because of blockage by different inhibitory proteins, including Bcl-2,
the presence of both Fas-receptor and Fas ligand in melanoma cells is
necessary but not sufficient to initiate death signaling. UVC-induced
degradation of Bcl-2 (Fig. 9D) correlates with the
progression of apoptosis seen in these cells. The elevated expression
level of FasL in LU1205/ATF2 cells may explain the higher level of
apoptosis seen after UV irradiation when compared with the apoptosis
seen in cells expressing the truncated form of ATF2.

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Fig. 9.
A-C, FasL promoter activity in
LU1205/neo and LU1205/ATF2 cells. The reporter constructs used are
indicated. D, Western blot analysis of FasL and Bcl-2
expression in LU1205/neo and LU1205/ATF2 cells following UVC treatment;
nonspecific band (ns) from Bcl-2 blot was used as a
reference band for protein loading.
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Melanoma cells are resistant to apoptotic signaling induced by TNF
,
which, as noted, elicits a survival signal in these cells. Since
NF-
B has been implicated in protection against TNF-mediated apoptosis (64-66), we next examined the possible effects of ATF2 overexpression on NF-
B DNA binding activity and transactivation before and after UVC treatment of melanoma cells. LU1205 cells contain
relatively high basal NF-
B activity (Fig.
10A). An additional increase
in DNA binding activity of NF-
B p65-p50 (Fig. 10B, the upper band of the specific NF-
B DNA-binding complex) and
transactivation of NF-
B were observed in ATF2- and
ATF2
1-195-transfected cells, both at the basal and at the
UVC-induced levels (Fig. 10, A and B). Increased
levels of NF-
B activity after UVC irradiation of ATF2-overexpressing
cells, when compared with the control cells, are due to down-regulation
of I
B
levels in these cells 18 h after UVC treatment (Fig.
10C), possibly as a result of ATF2-dependent regulation of I
B expression. Since UVC treatment causes prolonged activation and nuclear translocation of NF-
B (0.5-20 h) when compared with the short NF-
B activation in response to TNF
treatment (10-90 min), it is not possible to distinguish between
primary and secondary TNF-mediated activation of NF-
B in melanoma
cells. However, the abundance of NF-
B in these cells, especially in ATF2-overexpressing cells, is likely to represent an additional factor
that promotes Fas- versus TNF-mediated apoptosis of
late-stage melanoma cells.

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Fig. 10.
A, NF- B activity in ATF2-transfected
LU1205 cells. LU1205/neo, LU1205/ATF2, and LU1205/ATF2 1-195 cells
were transiently transfected with either 4xNF- B-CAT or with the
empty vector pBL4-CAT in the presence of pCMV- -gal. Two days after
transfection, cells were irradiated with UVC and protein extracts were
prepared 18 h later. The protein concentration of extracts was
normalized based on -gal activity. Relative CAT activity is
indicated. B, electrophoretic mobility shift assay of
nuclear extracts of control and LU1205/ATF2 cells 3 h after UVC
irradiation was performed as described previously (29). The positions
of NF- B DNA-binding complexes are indicated by arrows;
the upper bands represent the p65-p50
heterodimer, the intermediate band reflects the
p50-p50 complex, and the lower band, nonspecific
binding (ns). C, Western blot analysis of
I B levels in the respective melanoma cell lines 18 h after
UVC treatment.
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DISCUSSION |
The functional role of ATF2 in the regulation of cell death and
survival in human melanoma was evaluated by overexpression of its
full-size and truncated (without transactivation domain) forms. Here we
provide evidence that both forms of ATF2 serve as potent accelerators
of UVC-induced cell death in metastatic human melanoma LU1205 cells.
Apoptosis following UVC treatment and its enhancement by overexpression
of both forms of ATF2 were demonstrated by characteristic changes in
cell morphology, redistribution of phosphatidylserine in the plasma
membrane (annexin-V-FITC staining), activation of caspase cascade, PARP
cleavage, and DNA fragmentation.
Among the important determinants influencing the degree of
ATF2-mediated transactivation are: (i) alternative promoter usage of
the ATF2 gene and differential splicing, which create several ATF2
isoforms that exhibit different transcriptional output (full-length ATF2 in its native form is transcriptionally inactive as a result of
inhibitory intramolecular interaction, whereas a 42-kDa spliced form
(
150-248) is constitutively active; Refs. 33 and 44); (ii)
regulation of the transcriptional activity of ATF2, like other members
of the ATF/CREB and Jun/Fos families, by phosphorylation, including
JNK- and p38-directed phosphorylation of threonine residues at
positions 69 and 71 (39, 67- 69); (iii) ATF2 heterodimerization with
other proteins of the ATF/CREB and Jun/Fos families, which results in
varying degrees of transcriptional output (41, 46, 47) (ATF2
heterodimerization also plays an important role in its stability; Ref.
48); and (iv) interaction with the transcriptional coactivator p300
(dependent on phosphorylation of Ser121), which contributes
to ATF2 transcriptional activity (70). This tight regulation suggests
that any of these variables (hypophosphorylation, altered ratio between
different full-length and spliced forms, a change in the nature of the
heterodimers) may suppress ATF2-dependent transcription.
Indeed, silencing effects were described for different ATF2/CREB family
members. For example, binding of CREB and CREM to the AP-1 site
inhibited activation by c-Jun (71), ATF2 down-regulated hepatitis B
virus X promoter activity by competition for the AP-1 binding site and
formation of ATF2-Jun heterodimers (72), and CREB and ATF1 were shown
to be potent inhibitors of several transcription factors (73). The
dominant negative form of CREB efficiently reduced radiation resistance
(32), and inhibited tumor growth and metastatic potential of human
melanoma through squelching of other CREB-associated proteins (31, 74).
The relationship between changes in ATF2-dependent
transactivation after UVC irradiation and the degree of apoptosis, as
demonstrated in the present study, suggest that ATF2 tightly regulates
expression of genes that control apoptosis.
One of the common causes of cell death is down-regulation of expression
and decrease in the concentration within the medium of essential growth
factors, for example, death of the IL-2-dependent CTLL-2
cell line upon deprivation of IL-2 (75). Late-stage metastatic melanoma
cells are characterized by autocrine growth stimulation as a result of
production of several cytokines, including bFGF, IL-6, IL-8, and TNF
(59). Numerous observations indicate that TNF
promotes the
metastatic potential of melanoma cells (12-14) as well as of some
other tumors (10, 76-78). Down-regulation of c-KIT levels in melanoma
cells has been implicated in the enhancement of melanoma tumorigenicity
and metastasis (79). Although the effect of TNF
on negative
regulation of c-KIT expression in melanoma has not been determined, it
has been described for normal bone marrow progenitor cells and myeloid
leukemia cells (80, 81). In general, TNF
plays a dual role in the
regulation of cell death and survival. This cytokine is an essential
growth factor not only for metastatic, but also for normal, cells in
certain circumstances, e.g. in immature thymocytes (82). On
the other hand, this cytokine induces apoptosis of some tumor cell
lines and elicits an unusually wide range of biological responses
(7-9). Expression of the TNF
gene is regulated by different
transcription factors, including AP-1, ATF2, NFAT, Erg-1, C/EBP
, and
NF-
B (18-22). The present study demonstrated the ability to
modulate TNF
transcription using both full-length and truncated
forms of ATF2, resulting in increased susceptibility of UV-treated
melanoma cells to programmed death. The dominant negative form of c-Jun
(TAM 67) (53) also had a similar effect, further indicating that
down-regulation of TNF
transcription could be due to
heterodimerization of inactive ATF2 with c-Jun. In contrast,
overexpression of functionally active c-Jun allowed the negative effect
of ATF2 on TNF
expression to be overcome and partially rescued UV-
and ATF2-mediated apoptosis, suggesting that ATF2 silences the c-Jun
contribution to TNF
expression.
The present study also demonstrates that UVC-induced apoptosis of
LU1205 as well as ATF2-transfected LU1205 cells is mediated by Fas
death signaling. The role of Fas signaling in radiation-induced apoptosis was described in several normal and tumor cell systems (83-87). It was also demonstrated that UVC irradiation by itself may
initiate trimerization of the Fas receptor and induction of signaling,
substituting FasL (83, 87). However, it is still unresolved whether
UVC-induced Fas trimerization per se is sufficient to
mediate apoptosis, as it is difficult to exclude other cellular changes
elicited by UV irradiation. Melanoma cells expressing full-length ATF2
were found to express higher levels of FasL protein, which may also
contribute to Fas-mediated apoptosis, as was shown in UV-induced
apoptosis of human lymphocytes (63, 84). Elevated FasL expression may
explain the higher degree of apoptosis seen after UV irradiation of
melanoma cells that express full-length ATF2 versus
truncated form, as, in addition to silencing TNF, by both forms of
ATF2, only the full-length form appears capable of mediating positive
transcriptional signals. The ability of full-length ATF2 to elicit a
negative signal in the case of TNF
and a positive signal in the case
of FasL may be due to its heterodimerized partner, the overall
contribution of neighboring transcription factors on a given promoter,
and a possible requirement for the contribution of co-activators.
Clearly, the effects of full-length ATF2 as a transcription factor are
impaired due to its poor phosphorylation. We did not, however, complete
analysis of all spliced forms of ATF2 expressed in these melanoma cells
and thus cannot exclude further interplay between themselves and other
bZIP partners.
The resistance of cells against TNF-mediated killing is linked to
NF-
B expression (9, 64-66). Metastatic melanoma LU1205 cells, used
in the present study, are also characterized by moderate NF-
B
activity that may provide resistance against TNF
-mediated apoptosis.
Indeed, inhibition of NF-
B activity by I
B
transformed TNF
from a protective to a death
factor.2
Our data extend previous findings regarding down-regulation of
radioresistance in human melanoma MeWo cells by truncated forms of CREB
and ATF2 (32, 36). We have shown in the present study that the
truncated form of ATF2 increased UVC-induced apoptosis. Thus, an
elevated level of apoptosis coincides with reduced resistance to UV
irradiation. Surprisingly, comparison of ATF2
1-195 with full-length
ATF2 overexpression revealed that while both elicit equally efficient
silencing of TNF
expression, full-length ATF2 was more efficient in
elevating UV-mediated apoptosis, possibly because of its ability to
increase expression of FasL, which boosts death signaling in these
melanoma cells. This is unlike the effect of full-length ATF2 in early
stage melanoma cells DM3211, i.e. an increase radiation
resistance (36), suggesting that the impact of ATF2 on cell survival
depends on its proper activation and interaction with other factors
that have been altered during melanoma progression. Indeed, the balance
between various ATF2 forms, c-Jun, and other members of the CREB/ATF
and AP-1 families is expected to change during melanoma development as
was previously shown in the B16 mouse melanoma model (88).
In summary, we established an inverse correlation between TNF
expression as a survival factor and the consequences of Fas-mediated death signaling in melanoma cells. Our findings provide the foundation for design of ATF2-based reagents that could be useful in altering the
rate of irradiation-mediated programmed death of melanoma cells.