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Tumor Biology |
-Tocopheryl Succinate-induced Apoptosis of Human Breast Cancer Cells1
Division of Nutrition/A2703 [W. Y., Q. Y. L., F. M. H., B. G. S., K. K.] and School of Biological Sciences [W. Y., Q. Y. L., F. M. H., B. G. S.], University of Texas at Austin, Austin, Texas 78712
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ABSTRACT |
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 Top ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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-tocopherol succinate (vitamin E succinate, VES) is a potent,
selective apoptotic agent for cancer cells but not normal cells. VES has been shown to
inhibit the growth of a wide variety of tumor cells in cell culture and
animal models. Studies addressing mechanisms of action of VES-induced
apoptosis have identified transforming growth factor-ß,
Fas/CD95-APO-1, and mitogen-activated protein kinase (MAPK) signaling
pathway involvement. Here we show that MAPKs, the extracellular
signal-regulated kinases (ERK), and the stress-activated protein
kinases, c-Jun NH2-terminal kinases (JNK), but not p38, are critical
mediators in VES-induced apoptosis of human breast cancer MDA-MB-435
cells. VES activates ERK1/2 and JNK both in level and duration of
kinase activity. Expression of dominant negative mutants of ERK1,
MAPK/ERK activator-1, or JNK1 but not p38 blocked phosphorylation of
the substrate glutathione S-transferase-c-Jun and inhibited VES-induced
apoptosis. Increased phosphorylation and transactivation activity of
nuclear transcription factors c-Jun, ATF-2, and Elk-1 are observed
after VES treatments; however, only c-Jun and ATF-2 appear to be
involved in VES-induced apoptosis based on antisense blockage
experiments. Collectively, these results imply a critical role for ERK1
and JNK1 but not p38 in VES-induced apoptosis of human MDA-MB-435
breast cancer cells. |
INTRODUCTION |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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VES is a succinate ester of natural vitamin E (RRR--tocopherol) and
is used as a vitamin E source in some commercial supplements. Vitamin E
in blood and tissues is usually in the unesterified RRR--tocopherol
form; however, intact succinate esters of vitamin E can be taken up
directly by cells in culture and in vivo
(10, 11, 12, 13)
. VES is a member of a group of compounds
including retinoids and deltanoids (vitamin D metabolites and
analogues), tamoxifen (an antiestrogen), and monoterpenes that inhibit
the growth of tumor cells and have in common the ability to induce
tumor cells to secrete biologically active TGF-ß and to trigger
apoptosis (14, 15, 16, 17)
.
MAPKs represent a family of kinases that transduce diverse extracellular stimuli (mitogenic growth factors, environmental stresses, and proapoptotic agents) to the nucleus via kinase cascades to regulate proliferation, DNA synthesis arrest, differentiation, and apoptosis (18, 19, 20, 21, 22) . There are three well-defined MAPK pathways in mammalian cells: the ERK1/ERK2 cascade (18) ; and the stress-activated JNK (19 , 20) and p38 (20) MAPK cascades.
MAPKs are activated through phosphorylation of specific threonines
and tyrosines by dual specificity kinases via a four-step kinase
cascade: MAPK4 (MAP Kinase Kinase Kinase Kinase,
MKKKK/MEKKKs; TAB1); MAPK3 (MAP Kinase
Kinase Kinase, MKKK/MEKKs; Raf; TAK);
MAPK2 (MAP Kinase Kinase; MEKK/MEKs); and
MAPK1 (MAP Kinase; ERK1/ERK2, JNKs, and p38s).
Key targets of MAPKs include the nuclear transcription factors c-Myc,
Sap1, Elk-1, c-Jun, activating transcription factor-2, nuclear factor
B, Max, and MEF2 (18, 19, 20, 21, 22)
. Because MAPKs play
important roles in permitting cells to perceive and react to numerous
extracellular stimuli, it is not surprising that the MAPK cascades have
multiple substrates including shared, overlapping substrates,
cross-cascade interactions, and interconnections with other signal
transduction systems, all of which enable the MAPKs to mediate
coordinated responses but also make their study difficult because of
the high degree of complexity. Furthermore, MAPKs exhibit cell
type-specific and stimulus-specific responses (18)
.
Previous studies by our lab have documented the involvement of AP-1 in induction of apoptosis by VES in MCF-7 human breast cancer cells (23) . These studies demonstrated that VES treatment resulted in sustained, elevated levels of c-jun mRNA and c-Jun protein levels and increased AP-1 consensus element binding activity. Expression of a dominant negative interfering mutant of c-Jun in the MCF-7 human breast cancer cells partially protected the cells against VES-initiated apoptosis (23) . Studies in MDA-MB-435 cells supported the notion that c-Jun plays a critical but not totally sufficient role in VES-induced apoptosis, and implicates JNK activation in this process (24) .
To better define how VES induces apoptosis, we have additionally addressed the involvement of MAPKs. These data demonstrate: (1) VES treatment of human MDA-MB-435 breast cancer cells produces an early, transient activation of ERK1/2, a slightly later, prolonged activation of JNK1, but no activation of p38; (2) dominant negative mutants of ERK1 and JNK1, but not a dominant negative mutant of p38, can partially block VES-induced apoptosis; (3) VES increases the phosphorylation status and transactivation activity of nuclear transcription factors c-Jun, ATF-2, and Elk-1; and (4) antisense oligomers to c-Jun and ATF-2, but not antisense oligomers to Elk-1, can partially block VES-induced apoptosis. Taken together, these data support the hypothesis that ERK1 and JNK1 are critical yet not totally sufficient mediators of VES-induced apoptosis of human breast cancer cells.
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MATERIALS AND METHODS |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Western Immunoblot Analyses.
Whole-cell protein extracts were prepared as described previously
(8)
, and 100 µg of protein were loaded per lane,
separated using SDS-PAGE on a 10–12% gel under reducing conditions,
and electroblotted onto a nitrocellulose membrane (0.2 µM
pore Optitran BA-S-supported nitrocellulose; Schleicher and Schuell,
Keene, NH). Equal loading was verified using GAPDH antibody (produced
in house). Immunoblotting was performed using primary rabbit antibodies
with specificity for active (dually phosphorylated) ERK, JNK, or p38
(Promega, Madison, WI). Primary rabbit antibodies with specificity for
ERK1/2, JNK1, c-Jun, ATF-2, Elk-1, MEK1, HA-tag, and primary goat
antibody specific for p38 were purchased from Santa Cruz Biotechnology
(Santa Cruz, CA). Murine antibodies to the Flag-tag were purchased from
Sigma Chemical Co. Horseradish peroxidase-conjugated goat antirabbit or
goat antimouse secondary antibodies were purchased from Jackson
Immunoresearch Laboratory (West Grove, PA). Horseradish
peroxidase-conjugated donkey antigoat serum was purchased from Santa
Cruz Biotechnology. Immune complexes were visualized using enhanced
chemiluminescence detection (Pierce Chemical Co., Rockford, IL). Fold
differences in level of chemiluminescence were determined by
densitometric analyses.
DAPI Staining and Apoptotic Evaluation.
Apoptosis was assessed based on nuclear morphology using the
fluorescent DNA dye DAPI, as described previously (8)
.
Cells in which the nucleus contained clearly condensed chromatin or
cells exhibiting fragmented nuclei were scored as apoptotic.
Apoptotic data are reported as the percentage of apoptosis,
obtained by determining the number of apoptotic cells versus
the total number of cells. For each sample, a minimum of 3 counts
involving 
100–200 cells/count were scored. Apoptotic data are
presented as mean ± SD for three independent
experiments.
MAPK Activity Assays (Immune Complex Kinase Assays).
MAPK activity assays were conducted as described previously
(18)
. Briefly, whole cell lysates (100–200 µg/sample)
were first immunoprecipitated with antibodies specific for MAPKs
(ERK1/2, JNK1, or p38) before assaying for substrate phosphorylation.
Antibodies used included a rabbit antibody to ERK that recognized
primarily ERK1 but also detected ERK2, a rabbit antibody that
recognized JNK1, and a goat antibody that recognized p38 (Santa Cruz
Biotechnology). Immune precipitated MAPKs were reacted directly with 2
µg of GST-c-Jun (amino acids 1–79; a gift of Dr. Roger Davis,
Howard Hughes Medical Institute, University of Massachusetts Medical
School, Worcester, MA).
Assessment of Whole-Cell Kinase Activity Capable of
Phosphorylating Nuclear Transcription Factors c-Jun, ATF-2, and Elk-1.
In vitro kinase assays were conducted as described
previously (24)
. In addition to the GST-c-Jun 1–79(1–79)
substrate described above, GST-ATF-2 1–109(1–109), and GST-Elk-1 (307–428;
gifts from Dr. Roger Davis) were used for these studies.
c-Jun-, ATF-2-, and Elk-1-Transactivation Activity Assay.
c-Jun-, ATF-2-, and Elk-1-transactivation activities were determined as
described previously (24)
. Briefly, MDA-MB-435 cells were
cotransfected with a chimeric GAL4 DNA-binding domain construct coupled
with the transactivation domains of c-Jun 1–79(1–79), AFT-2 1–109(1–109), or
Elk-1 plus the pGSE1bLuc reporter construct, which contains five GAL-4
sites cloned upstream of a minimal promoter element and the firefly
luciferase gene (kind gifts from Dr. Roger Davis) plus a
ß-galactosidase plasmid for normalization of transfection
efficiencies. Light units were determine for luciferase expression, and
absorbance was determined for ß-galactosidase activity. Data are
presented as relative light units, which is based on the ratio of
luciferase light units to ß-galactosidase absorbance units.
Transient Transfection of Cells with DN ERK1, MEK1, JNK1, and p38
Constructs.
Constructs containing dominant negative acting mutants of ERK1 and MEK1
were gifts from Dr. Jacques Pouyssegur (Center de Biochimine, Center
National de la Recherche Scientifique, Universite de Nice, Nice,
France; 26, 27, 28
). Constructs containing dominant negative
acting mutants of JNK1 and p38 were gifts from Dr. Roger Davis
(29)
.
DN ERK1 [pcDNA expression (CMV promoter) vector encoding HA/p44T192Amapk] has threonine 192 replaced with alanine (27 , 28) and DN MEK1 (pECE-HA/S222AMEK1) has amino acid serine 222 replaced with alanine (26) . DN-JNK1 [pcDNA3-Flag-JNK1(APF)] has the tyrosine 185 and threonine 183 amino acids, which require phosphorylation for activity replaced with alanine and phenylalanine, respectively. DN p38 [pCMV-Flag-p38(AGF)] has the tyrosine 182 and threonine 180 amino acids, which require phosphorylation for activity replaced with alanine and phenylalanine, respectively (29) . DN JNK1 and DN p38 contain the Flag epitope (-Asp-Try-Lys-Asp-Asp-Asp-Asp-Lys-), and DN ERK1 and DN MEK1 contain a sequence coding for a nine-residue immunodominant peptide from influenza virus hemagglutinin, which permits detection by immunoblotting using reagents specific for the Flag-tag and the HA-tag, thus allowing discrimination of DN proteins from endogenous proteins.
Transient transfection of cells with expression plasmids coding for DN-interfering proteins was conducted using LipofectAMINE and Plus Reagent (Life Technologies, Inc.) following the instructions of the manufacturer. Briefly, MDA-MB-435 cells at 1.5 x 105/well in 12-well plates were used for apoptosis assessment, and cells at 1.6 x 106/T-25 flask were used for Western immunoblot analyses. The cells were allowed to adhere overnight, then washed twice with serum-free medium (MEM) and incubated for 3–4 h with either 0.5 ml of MEM-Option serum-free medium (Life Technologies, Inc.) containing 100 µl of plasmid/Plus reagent/LipofectAMINE complex for 12-well plates or 3 ml of MEM-Option serum-free medium containing 700 µl of plasmid/Plus reagent/LipofectAMINE complex for T-25 flasks. The plasmid/Plus reagent/LipofectAMINE complex was made by mixing 0.7 µg of plasmid/50 µl of serum-free medium with 5 µl of Plus reagent followed by 15 min incubation, then mixing the reaction with 4 µg of LipofectAMINE Reagent/50 µl of serum-free medium followed by another 15 min incubation. The cells were cultured in normal culture medium for 20 h followed by VES- or VEH-treatments for different time periods.
Transient transfection efficiency in MDA-MB-435 cells with Flag-tagged
DN JNK was 
50%, as determined by immunohistochemical staining using
mouse anti-Flag primary antibody, antimouse horseradish
peroxidase-labeled secondary antibody, and substrate (data not shown).
Briefly, 20 h after transfection with either Flag-tagged DN JNK or
vector control, cells (1.5 x 105/well in 12-well plates) were washed with PBS
twice then fixed with 4% paraformaldehyde for 20 min at 4°C. After
fixation, cells were washed with PBS twice, pretreated with PBS
containing 1% FCS for 10 min at room temperature, then incubated with
2 µg/ml anti-Flag antibody (Sigma Chemical Co.) in PBS containing 1%
FCS for 1 h at 4°C. The cells were washed with PBS twice then
reacted with secondary antibody and peroxidase substrate contained in
the Vectastain ABC Kit (Vector Laboratories, Inc., Burlingame, CA)
following the manufacturer’s instructions.
Stable Transfection of Cells with DN JNK1-inducible (TET-on)
Construct.
MDA-MB-435 cells were stably transfected with a TET-on-inducible
expression plasmid (Clontech) and TRE-Flag-JNK1(APF) plasmid encoding
DN JNK1(APF) described above [produced in-house using the Clontech
system (K1620–1)]. To generate inducible clones, MDA-MB-435 cells
were first transfected with the pTet-On vector, and stable clones were
selected by growing the cells in the presence of 0.5 mg/ml of G418
(Sigma Chemical Co.) as a selective antibiotic, followed by
transfection with pTRE DN JNK1(APF) and selection of stably transfected
clones using 0.5 mg/ml of G418 and 0.2 mg/ml of hygromycin B (Clontech)
as selective antibiotics. Transfections were performed using
LipofectAMINE Plus Reagent described as above. Inducible clones were
screened by Western immunoblot analyses to determine levels of
endogenous JNK1 and Flag-tagged DN JNK1 expression after 2 µg/ml of
doxycyline treatment for 2 days.
Antisense Knockout of c-Jun, ATF-2, and Elk-1.
Phosphothiorate-modified antisense and sense DNA oligonucleotides
to c-Jun (antisense, CGT TTC CAT CTT CGT AGT CAT and sense, ATG ACT GCA
AAG ATG GAA ACG), ATF-2 (antisense, CAC ATG TAA CTT GAA TTT CAT and
sense, ATG AAA TTC AAG TTA CAT GTG), and Elk-1 (antisense, CAG CGT CAC
AGA TGG GTC CAT and sense, ATG GAC CCA TCT GTG ACG CTG) were purchased
from Operon Technologies (Alameda, CA) and transfected into MDA-MB-435
cells using LipofectAMINE (Life Technologies, Inc.) as described
previously (24)
and as described above without the Plus
reagent. Briefly, the cells were transfected with 0.5 ml of MEM-Option
serum-free medium (Life Technologies, Inc.) plus 100 µl of
oligomer/LipofectAMINE complex for 12-well plates and 3 ml of
MEM-Option serum-free medium plus 700 µl of oligomer/LipofectAMINE
complex for T-25 flasks. The oligomer/LipofectAMINE complex was made by
mixing 2 µg oligomers/50 µl MEM-Option serum-free medium with 8
µg of LipofectAMINE/50 µl MEM-Option serum-free medium for 45 min.
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RESULTS |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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, top panel). Levels of ERK1/2 proteins were not increased
by VES treatment (Fig. 1A
, bottom panel).
Densitometric analyses showed peak ERK1 (p44) activation at 2 h to
be 5-fold higher than at 1 h after VES treatment, whereas peak
ERK2 (p42) activation at 2 h was 17-fold higher than the VEH
control. VES induced a prolonged JNK1 activation starting at 1.5 h
and continuing through 24 h after treatment initiation (Fig. 1B
, top panel). Levels of JNK1 were not increased with VES
treatment (Fig. 1B
, bottom panel). Densitometric analyses
showed JNK activation at 12 h after VES treatment to be 10-fold
higher than JNK activity after treatment with VES for 1.5 h. VES
treatment did not activate p38 (Fig. 1C
, top panel).
MDA-MB-435 cells treated with 20 µM AA for 15
min were used as positive controls for activation of p38. p38 protein
levels were not modulated by VES treatment (Fig. 1C
, bottom
panel).
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, top panel). Densitometric analyses showed peak
ERK-mediated phosphorylation of GST-c-Jun at 2 h after VES
treatment to be 4.4-fold higher than phosphorylation of substrate after
VEH treatment. JNK1 immunoprecipitated from VES-treated cells was
capable of phosphorylating GST-c-Jun as early as 3 h after
treatment, and JNK kinase activity remained active for 12 h after VES
treatment (Fig. 2B
, top panel). Densitometric
analyses showed the peak JNK activity for the GST-c-Jun substrate at
12 h after VES treatment to be 2.3-fold higher than kinase
activity of the VEH control. Phosphorylation of GST-c-Jun by p38
immunoprecipitated from VES-treated cells did not increase with
treatment time and did not differ from the VEH control (data not
shown). The increased kinase activity of ERK1/2 or JNK1 is not
attributable to enhanced protein levels, because the levels of ERK1/2
and JNK proteins did not change after VES treatment (Fig. 2, A and B
, bottom panels, respectively).
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To evaluate the relevance of ERK activation in VES-induced apoptosis,
MDA-MB-435 cells were transiently transfected with an expression
construct containing dominant negative ERK1 (T192A) or DN MEK1 (S222A),
a direct upstream activator of ERK. DN ERK1 blocked VES-induced
apoptosis produced by treating the cells with either 10 or 20 µg/ml
VES for 2 days by 45 and 49%, respectively (Fig. 3A)
. Likewise, DN MEK1 blocked VES-induced apoptosis by 42 and
43% (Fig. 3B)
. Furthermore, both DN ERK1 and DN MEK1
blocked: (a) VES-induction of active ERK1/2 (Fig. 3C
,
top panel); and (b) VES-induction of ERK kinase
activity (as measured by the ability of immunoprecipitated ERK1/2 to
phosphorylate GST-c-Jun; Fig. 3C
, second panel).
Densitometric analyses showed that DN ERK1 in comparison to vector
control reduced levels of active ERK1 by 48% and decreased
phosphorylation of GST-c-Jun by 43%. Likewise, densitometric
analyses showed that DN MEK1 in comparison to vector control reduced
levels of active ERK1 by 68% and active ERK2 by 48% and
decreased phosphorylation of GST-c-Jun by 59%. Verification that
the MDA-MB-435 cells were expressing DN ERK1 and DN MEK1 comes from
Western immunoblot analyses showing three bands (endogenous ERK1/2 and
HA-tagged DN ERK1) in cells transfected with DN ERK1 versus
two bands (endogenous ERK1/2) in empty vector control cells (Fig. 3C
, third panel) and two bands (endogenous MEK1 and
HA-tagged DN MEK1) in cells transfected with HA-tagged DN MEK1,
versus 1 band (endogenous MEK1) in empty vector control
cells (Fig. 3C
, fourth panel). Additional evidence of
successful transfection was documented by analyses of whole cell
extracts using antibodies to the HA epitope of the DN ERK1 and MEK1
proteins (Fig. 3C
, fifth panel). Levels of GADPH were used
to verify lane loads (Fig. 3C
, sixth panel).
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. Levels of endogenous JNK + DN Flag-tagged JNK
expressed by the transfected cells are depicted in Fig. 4B
, top
panel. Levels of Flag-tagged DN JNK expressed are depicted in Fig. 4B
, middle panel. GADPH levels served to help verify lane
loads (Fig. 4B
, bottom panel). The ability of whole cell
extracts from DN JNK-transfected cells to inhibit VES-mediated
phosphorylation of GST-c-Jun, in comparison to VES-treated empty vector
control cells, is depicted in Fig. 4B
, third panel.
Densitometric analyses showed that DN JNK, in comparison to vector
control, decreased phosphorylation of GST-c-Jun by 53%. Transient
transfection efficiency in MDA-MB-435 cells with Flag-tagged DN JNK was
50% as determined by color reaction using mouse anti-Flag primary
antibody, antimouse horseradish peroxidase-labeled secondary antibody,
and substrate (Vectastain ABC Kit; Vector Laboratories; data not
shown).
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. Although high levels of Flag-tagged protein were
expressed in cells transiently transfected with DN p38 (Fig. 4B
,
second panel) there was no reduction in phosphorylation of
GST-c-Jun by whole cell extracts from the DN p38-transfected cells
(Fig. 4C
, third panel).
To additionally confirm the role of JNK in VES-induced apoptosis, yet
another approach, namely the establishment of stable clones of
MDA-MB-435 cells with inducible DN JNK(APF), was used. MDA-MB-435 cells
were stably transfected with an inducible (Tet-on) Flag-tagged DN JNK
construct, and clones expressing high levels of DN JNK after 2 days of
treatment with 2 µg/ml of Dox were chosen (three such clones
designated 19, 35, and 53 are reported here). For experiments,
Dox-induced and noninduced cells were treated with 20 µg/ml of VES
for 2 days. Induction of DN JNK in clones 19, 35, and 53 by Dox reduced
the ability of VES to increase the phosphorylation status of GST-c-Jun
(Fig. 5A
, top panel). Densitometric analyses of these
data showed 2.4-, 1.4-, and 1.8-fold reductions in comparison to levels
of GST-c-Jun substrate phosphorylation after VES-treatment of
noninduced cells for clones 19, 3, and 53, respectively. JNK1
(endogenous + DN JNK1) protein levels expressed in induced
(+) clones 19, 35, and 53 were 2.0-, 3.2-, and 6.4-fold higher than JNK
(endogenous only) levels expressed by noninduced (-) clones (Fig. 5A
, lower panel). In comparison to VES-treated noninduced
cells from clones 19, 35, and 53, VES-induced apoptosis of Dox treated
clones 19, 35, and 53 was reduced by 52, 42, and 49%, respectively
(Fig. 5B)
.
|
. Densitometric analyses of the 12 h data showed peak
phosphorylation for c-Jun, ATF-2, and Elk-1 to be 3.5-, 10.2-, and
5.3-fold higher in the VES versus VEH control samples,
respectively.
|
. VES treatments for 12 or 24 h
produced increases in luciferase activities of 1.9- and 3.6-fold for
c-Jun-, 2.0- and 5.3-fold for ATF-2, and 4.0- and 6.3-fold for
Elk-1-mediated transactivation in comparison with the levels of
transactivation measured in VEH control-treated cells (Fig. 6B)
. Cells cotransfected with a control GAL4 DNA-binding
domain construct containing no transactivation domain plus the
luciferase reporter construct exhibited low levels of luciferase
activity after either VES or VEH treatment (data not shown).
Antisense Oligomers to c-Jun and ATF-2 but not to Elk-1 Reduce
VES-induced Apoptosis.
Because previous studies by our lab had shown that c-Jun is critical to
VES-induced apoptosis (24)
, it was of interest to
determine whether or not either ATF-2 or Elk-1 might be playing a
necessary role in VES-induced apoptosis, so we conducted functional
knockout experiments using antisense oligonucleotides. Repeat analyses
of antisense oligonucleotide functional knockout of c-jun
were performed for comparative purposes. MDA-MB-435 cells transiently
transfected with antisense or sense oligomers to c-Jun,
ATF-2, and Elk-1 were cultured with 20 µg/ml of
VES for 2 days. Antisense oligomers to c-Jun and
ATF-2, in comparison to cells transfected with corresponding
sense oligomers, blocked VES-induced apoptosis by 49 and 39%,
respectively (Fig. 7A)
. VEH-treated cells gave basal levels (<5%) of apoptosis
(data not shown). In contrast, antisense oligomers to Elk-1
had little to no effect on VES-induced apoptosis (Fig. 7A)
.
Evidence for transfection efficiency and functionality of the antisense
oligomers to c-jun, ATF-2, and Elk-1
is provided by Western immunoblot analyses, demonstrating that cells
transiently transfected with antisense oligomers, in comparison to
sense oligomers, exhibited reduced expression of c-Jun, ATF-2, and
Elk-1 (Fig. 7B
, top panel). Densitometric analyses showed a
3.9-, 2.3-, and 2.7-fold reduction, respectively. GAPDH levels were
used as lane load controls (Fig. 7B
, bottom panel).
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DISCUSSION |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Studies presented here document that VES-triggered apoptosis involves
ERK1, MEK1, and JNK1 but not p38. Both ERK1/2 and JNK1 were activated
after VES treatment as documented by detection of the active
(phosphorylated) forms of these kinases using antibodies specific for
the active enzyme as well as kinase activity assays using
immunoprecipitated kinases and using GST-c-Jun as substrate.
Furthermore, transient expression of dominant-negative mutants of ERK1
and JNK1 (also stably transfected inducible DN JNK) blocked VES-induced
phosphorylation of GST-c-Jun substrate as well as blocking VES-induced
apoptosis by 50%, indicating that activated ERK1 and JNK1 are
involved in VES-induced apoptosis. Transient transfections with DN p38
had no effect on either phosphorylation of GST-c-Jun substrate or
apoptosis induced by VES. Thus, collectively, our results indicate a
critical role for ERK1 and JNK1 but not p38 in VES-induced apoptosis of
human MDA-MB-435 cells.
The duration of JNK activation is important in determining cell fates
(30)
. Mitogenic stimuli produce transient JNK
induction, whereas environmental stresses and apoptotic triggering
agents produced sustained JNK activation (30)
. Studies
reported here and previous studies by our lab document that JNK levels
are elevated for a prolonged time period after VES treatment
(24)
. VES treatments induce activation of JNK1 beginning
1.5 h after VES treatment and produce a sustain elevation for
24 h with peak levels at 12 h after treatment involving a
10-fold increase over control values. The kinetics of JNK1 but not
ERK1/2 activation correlate with the kinetics of increased
phosphorylation of c-Jun and ATF-2, which appear to play a role in
VES-induced apoptosis and correlate with the onset of early VES-induced
apoptotic alterations such as activation of caspase 3 and cleavage of
poly(ADP-ribose) polymerase, which occur 12 h after VES
treatment.4
Because either JNK or p38 or both JNK and p38 can be activated by
various stressors, it is of interest that VES activates JNK but not
p38. To rule out the possibility that this differential activation
might be attributable to loss of functionality of the p38
stress-activated protein kinase in this neoplastic cell line, we
treated MDA-MB-435 cells with an agent (AA), which is reported in the
literature to be capable of activating p38 in human breast cancer cells
(31)
and showed that indeed the MDA-MB-435 cells used in
these studies have an activatable p38 (Fig. 1C)
.
The functional relevance of ERK1 activation to VES-induced apoptosis
was a surprise, because typically, activation of the ERK pathway is
associated with responses to mitogenic stimulation (18)
,
and the kinetics of ERK1 activation do not appear to correlate with
other apoptotic-related events in VES-treated cells. Although the
majority of ERK stimuli induce cell proliferation or differentiation,
it appears that some stimuli, for example, ligation of the cell surface
receptor Fas (CD95/APO-1) by its specific ligand or by anti-Fas
antibodies in human neuroblastoma cells, may use the ERK pathway to
trigger apoptosis (32)
. Furthermore, Mulder et
al. (33
, 34)
have reported the involvement of ERK2
(and JNK) in TGF-ß-mediated negative-growth control of human breast
cancer cells that retain responsiveness to TGF-ß. It should be noted
that the increase in ERK2 induced by TGF-ß was detected 10 min after
treatment initiation, was sustained for at least 30 min, and achieved a
level 1.7-fold above control values. This is a very different profile
from what we observe with VES treatments, which induce activation of
ERK1/2 beginning 1 h after VES treatment and produce a sustained
elevation for 3–4 h with peak levels 2 h after treatment
involving a 17-fold increase over control values. Thus, the duration
and intensity of ERK activation appears to be important in determining
cell fate, namely, growth arrest versus apoptosis. Both the
upstream activators of MEK1 and the downstream substrates of ERK1/2
that are playing a role in VES-induced apoptosis remain to be
identified.
Previous studies by our lab have shown that the transcription factor c-Jun is involved in VES-induced apoptosis of human breast cancer cells (23 , 24) . Data reported here show that in addition to c-Jun, ATF-2 but not Elk-1 is involved in VES-induced apoptosis. Studies by Dam et al. (35 , 36) have shown that c-Jun/ATF-2 dimers are major regulators of the c-Jun promoter. Previous studies by our lab have shown that VES induces prolonged and enhanced expression of c-Jun mRNA, protein, and AP-1 activity (23 , 24) . VES-mediated increases in c-jun mRNA occur at the transcriptional level (24) , suggesting that perhaps dual activation of c-Jun and ATF-2 may contribute to this event. Unlike c-Jun, the protein levels of ATF-2 are not modulated by VES. Because antisense oligomers to c-Jun can block VES-induced apoptosis, one possible mechanism whereby antisense oligomers to ATF-2 may block VES-induced apoptosis is via reduction of c-Jun protein levels.
We have shown previously that VES treatment of human breast cancer cells converts the cells from a nonresponsive to a responsive phenotype in regard to TGF-ß signaling and agonistic anti-Fas antibody stimuli (1 , 8) . VES-treated MDA-MB-435 cells secrete biologically active TGF-ß ligand and exhibit enhanced cell surface membrane levels of TGF-ß type II receptors (6) . Furthermore, VES treatment of MDA-MB-435 cells induces the translocation of cytosolic Fas to the cell surface membrane (8) . Blockage of either the TGF-ß or Fas signaling pathways with neutralizing antibodies decreases VES-induced activation of c-Jun kinases and decreases VES-induced apoptosis, suggesting the involvement of both pathways in these VES-mediated events (data not shown). Fas/FADD/caspase 8 signaling (37) does not appear to be occurring in VES-restored Fas signaling, rather, preliminary data suggest that Fas is activating JNK via Daxx. Likewise, preliminary data suggest that VES-restored TGF-ß signaling does not involve Smads but rather that TGF-ß activation of JNK may be via Rho/Cdc 42 and TAB/TAK-1 signaling (38, 39, 40) . Clarification of the contributions of these signaling pathways to VES-induced apoptosis will require additional study.
In summary, the overall goal of our studies is to characterize the signaling pathways producing VES-induced apoptosis, cellular differentiation, and cell growth inhibition. Expectations are that these studies will increase basic knowledge about fundamentally important signaling pathways in cancer cells, which will lead to the development of specifically targeted drugs to achieve improved cancer cell elimination. Studies reported here implicate MAPKs ERK1 and JNK1 but not p38, as well as transcription factors c-Jun and ATF-2 but not Elk-1 as being involved in VES-induced apoptosis.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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1 Supported by grants from the National Cancer
Institute (CA59739) and the Foundation for Research. 
2 To whom requests for reprints should be
addressed, at Division of Nutrition/A2703, University of Texas, Austin,
TX 78712-1097. Phone: (512) 471-8911; Fax: (512) 232-7040; E-mail: k.kline{at}mail.utexas.edu
3 The abbreviations used are: VES, vitamin E
succinate (RRR--tocopheryl succinate); AA, arachidonic acid; MAPK,
mitogen-activated protein kinase; AP-1, activator protein-1; ATF-2,
activating transcription factor-2; DAPI, 4',6-diamidino-2-phenylindole;
Dox, doxycycline; DN, dominant negative mutants capable of binding
substrate but incapable of activation by phosphorylation; Elk-1, ETS
domain protein; ERK, extracellular signal-regulated kinase; Fas,
CD95/APO-1; GAL4, yeast transcription factor involved in regulation of
galactose; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; GST,
glutathione S-transferase; GST-c-Jun/GST-ATF-2/GST-Elk-1, critical
phosphorylation protein domains of transcription factors c-Jun, ATF-2,
and ELK-1 fused to GST; JNK, c-Jun NH2-terminal kinase also called
stress-activated protein kinase; MEK, MAPK kinase; MDA-MB-435, estrogen
nonresponsive human breast cancer cells; p38, HOG1 kinases; TGF-ß,
transforming growth factor-ß; UT, untreated; VEH, vehicle.
4 Weiping Yu, Bob G. Sanders, and Kimberly Kline,
unpublished data.
Received 3/12/01. Accepted 6/21/01.
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