From the Department of Dermatology,
§ Department of Pediatrics and the H. B. Wells Center for
Pediatric Research, and the Departments of
** Pharmacology and Toxicology and
Biochemistry and
Molecular Biology, Indiana University School of Medicine,
Indianapolis, Indiana 46202
Received for publication, November 5, 2002, and in revised form, February 21, 2003
![]() |
ABSTRACT |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Most chemotherapeutic agents exert their
cytotoxic effects in part through the induction of apoptosis. In
addition, many chemotherapeutic agents are potent pro-oxidative
stressors. Although the lipid mediator platelet-activating factor (PAF)
is synthesized in response to oxidative stress, and many epidermal
carcinomas express PAF receptors, it is not known whether
PAF is involved in chemotherapeutic agent-induced
apoptosis. These studies examined the role of the PAF system
in chemotherapy-mediated cytotoxicity using model systems created by
retroviral mediated transduction of the PAF receptor-negative human
epidermal carcinoma cell line KB with the human PAF receptor (PAF-R)
and ablation of the endogenous PAF-R in the carcinoma cell line HaCaT
with a retroviral mediated inducible antisense PAF-R vector. The
presence of the PAF-R in these models resulted in an augmentation of
apoptosis induced by chemotherapeutic agents etoposide and mitomycin C
but not by tumor necrosis factor-related apoptosis-inducing
ligand or by C2 ceramide. Oxidative stress and
the transcription factor nuclear factor Apoptosis, or programmed cell death, is a fundamental
physiological process enabling the removal of damaged or infected cells and the control of cell populations (1). Apoptosis can occur during embryogenesis, induction and maintenance of immune tolerance, development of the nervous system, and endocrine-dependent
tissue atrophy (2). In addition to normal physiological conditions, essentially all chemotherapeutic agents exert their effects by induction of apoptosis (3). Thus, regulation of apoptosis can have
important consequences both during development and in the treatment of cancer.
Accumulating evidence suggests platelet-activating factor
(PAF1; 1-alkyl-2-acetyl
glycerophosphocholine)-mediated pathways are involved in cutaneous
inflammation and keratinocyte stress responses. PAF is a
glycerophosphocholine-derived lipid mediator implicated in numerous
inflammatory processes (for a review, see Ref. 4). Keratinocytes
synthesize PAF and related 1-acyl-PAF-like species in response to
various stimuli including ionophores, growth factors, PAF agonists, the
pro-oxidative stressor tert-butyl hydroperoxide, ultraviolet
light irradiation, or acute thermal damage (5-9). Although PAF can be
metabolized to other biologically active lipids, the majority of PAF
effects appear to be mediated by interaction with a G protein-coupled
receptor (GPCR), the PAF receptor (PAF-R) (for a review, see Ref. 10).
In addition to producing PAF, keratinocytes and many carcinoma cell
lines also express the PAF-R (11). Activation of the epidermal PAF-R
leads to the production and release of PAF, IL-6, IL-8, IL-10, tumor
necrosis factor- In addition to a tightly coupled enzymatic pathway for PAF synthesis
involving the subsequent actions of phospholipase A2 and
acetyltransferase, PAF and PAF-like lipids can be produced via direct
free radical-mediated cleavage of glycerophosphocholines (GPCs)
containing unsaturated fatty acids (e.g. arachidonate) at
the sn-2 position (15). Recent studies have suggested that the PAF-R is a target for ultraviolet B radiation in part through this
non-enzymatic pathway of PAF agonist formation (7, 8, 13, 14). For
example, the presence of the PAF-R augments the production of cytokines
IL-8 and tumor necrosis factor- Inasmuch as other proapoptotic agents including chemotherapeutic agents
are also potent pro-oxidative stressors, the objective of these studies
was to assess whether the epidermal PAF-R could augment the cytotoxic
effects of chemotherapeutic agents. Using a PAF-R-negative human
carcinoma cell line transduced with the PAF-R and a novel retroviral
mediated antisense strategy to ablate endogenous PAF-R expression in a
PAF-R-positive epithelial carcinoma cell line, we demonstrate that the
PAF-R can augment apoptosis due to etoposide and mitomycin C but not
other agents such as C2 ceramide and TRAIL.
The mechanism of how PAF-R activation could augment
chemotherapy-induced apoptosis was also examined in these studies, and it was found to be dependent upon activation of NF- Reagents--
All chemicals were obtained from Sigma unless
indicated otherwise. Recombinant human TRAIL/APO2L was purchased from
Chemicon (Temecula, CA). IL-1 Cell Culture--
The human epidermoid cell line KB and human
keratinocyte cell line HaCaT were grown in Dulbecco's modified
Eagle's medium (Invitrogen) and were supplemented with 10% fetal
bovine serum (Hyclone, Logan, UT). A KB PAF-R model system was created
by transduction of PAF-R-negative KB cells with the MSCV2.1 retrovirus
encoding the human leukocyte PAF-R as described previously (12). KB
cells transduced with the PAF-R (KBP) or with control MSCV2.1
retrovirus (KBM) were characterized by Southern and Northern blot
analysis and by radioligand binding and calcium mobilization studies to demonstrate that the PAF-R was functional (12). Similarly, to create a
KB cell line expressing the fMLP receptor (KBF), the fMLP-R cDNA
was cloned into the MSCV2.1 retroviral vector. The presence of a
functional fMLP-R in KBF cells was confirmed by Northern blotting to
demonstrate fMLP-R mRNA and by a positive intracellular calcium
mobilization response to exogenous fMLP using the fluorescent dye
Indo-1 as described previously (12). All experiments were replicated
with at least two separate KBM, KBP, and KBF clones.
Generation of HaCaT Cells Expressing an Inducible PAF-R Antisense
HaCaT as-PAFR System--
The HaCaT as-PAFR model system was
established using the RevTet-on system (Clontech).
PAF receptor cDNA was cloned in a reversed orientation into the
HindIII site of the response retroviral vector pRevTRE. The
insert orientation was confirmed by restriction mapping and sequencing.
To generate the retroviruses, amphotropic packaging cell line Phoenix
293 was transfected with either the retroviral Tet-on regulator, the
pRevTRE-as-PAFR, or the pRevTRE backbone using FuGENE 6 (Roche Applied
Science), and transient supernatants containing infectious amphotropic
retrovirus were collected 48 h later. In the first round of
infection, the infectious supernatant made from the retroviral Tet-on
regulator was used to infect the parental HaCaT cells, and cells
resistant to 1 mg/ml G418 were subjected to infection with the
infectious supernatant made from the pRevTRE-as-PAFR or the viral
backbone pRevTRE and selected with 600 µg/ml hygromycin. To increase
the number of gene copies in the cells, we performed a second round of
infection in which the transduced HaCaT cells that were double
resistant to G418 and hygromycin were infected with both viruses again.
Calcium mobilization studies and IL-8 biosynthesis studies were
performed as described previously (12). Radioligand binding studies
with PAF-R antagonist [3H]WEB 2086 were conducted as
described previously (12). Briefly, cells were plated in triplicate in
24-well dishes and treated with 10 nM [3H]WEB
2086 (PerkinElmer Life Sciences) ± 1 µM PAF. After
a 16-h incubation at 4 °C, the cells were washed and solubilized
with 1% Triton X-100, and radioactivity was measured using a
scintillation counter. Specifically bound [3H]WEB 2086 was defined as the difference between the binding of the radioligand
alone and in the presence of 1 µM PAF.
Generation of KBP Cells Expressing the Super-repressor
I Cell Death Detection Assay--
Apoptosis was determined
quantitatively using a cell death detection enzyme-linked immunosorbent
assay (Roche Applied Science) according to the manufacturer's
instructions. The kit measures the enrichment of mono- and
oligonucleosomes released into the cytoplasm of apoptotic cells as a
result of DNA degradation. The absorption was measured at 405 nm using
a microplate reader (Molecular Devices, Sunnyvale, CA). The enrichment
factor was calculated using the following formula: absorbance of
apoptotic cells/absorbance of control cells.
Caspase-3 Assay--
The activation of the caspase proteolytic
cascade was measured by the direct assay of caspase-3 enzyme activity
in cell lysates using a synthetic fluorogenic substrate (caspase-3
substrate, Ac-Asp-Glu-Val-Asp-AMC (Ac-DEVD-AMC); Alexis Biochemicals,
San Diego, CA) as described previously (25). Reactions were performed for 1 h at 37 °C. Release of the fluorogenic AMC moiety was
measured using a Hitachi F2000 spectrophotofluorometer (excitation, 380 nm; detection, 460 nm). The fluorescent intensity was converted to pmol
of AMC released by comparison to standards of AMC (Molecular Probes,
Eugene, OR). The specific activity of caspase-3 in cell lysates was
determined following quantitation of the total protein in the cell
lysates (NanoOrange protein quantitation reagent, Molecular Probes).
Intracellular Hydrogen Peroxide Measurements--
To measure
intracellular hydrogen peroxide levels, cells that were plated on
coverslips were loaded with a 10 µM concentration of the fluorescent dye CM-DCFDA (Molecular Probes) for 6 h at 37 °C in the dark. CM-DCFDA fluorescence was measured at an
excitation wavelength of 480 nm and an emission wavelength of 520 nm
using a Hitachi F2000 spectrophotofluorometer.
NF- The KB PAF-R Model System--
Since PAF may have both
receptor-dependent and -independent effects (secondary to
the formation of biologically active metabolites), our laboratory has
previously created a model system by transduction of the PAF-R into a
PAF-R-deficient epidermal cell line to study the role of the PAF-R in
epithelial cell biology. The human epidermal carcinoma cell line KB
does not express functional PAF-Rs unlike normal human keratinocytes
and the human keratinocyte-derived carcinoma cell line HaCaT (11, 12).
A PAF-R-positive KB cell line, KBP, was created by transducing KB cells
with a replication-deficient MSCV2.1 retrovirus containing the human
PAF-R cDNA. KB cells were also transduced with the retrovirus
backbone alone to establish a vector control cell line, KBM. Expression
of the PAF-R protein was verified by binding studies using radiolabeled
PAF-R antagonist WEB 2086 (12). Calcium mobilization studies
demonstrated that the KB PAF-R was functionally active (12). Therefore,
this in vitro epidermoid system consists of both
PAF-R-negative (KBM) and -positive (KBP) cells.
Cytotoxic Effects of Chemotherapeutic Agents in KB Cells--
In
initial experiments, the dose-response (Fig.
1) and time-response (Fig. 2) effects of
etoposide, mitomycin C, C2 ceramide, and TRAIL on apoptosis
in KBM versus KBP cells were determined by measurement of
caspase-3 enzyme activity. Exposing KB
cells to both chemotherapeutic agents etoposide and mitomycin C as well as C2 ceramide and TRAIL resulted in increased caspase-3
enzyme activity levels. The levels of caspase-3 enzyme induction were enhanced in PAF-R-expressing KBP over control KBM cells in response to
etoposide and mitomycin C. However, C2 ceramide and TRAIL
treatment resulted in similar levels of apoptosis in KBP and KBM cells. We next examined the effect of chemotherapeutic agents on KB cells using a cell death detection enzyme-linked immunosorbent assay as a
second distinct marker of apoptosis in carcinoma cells. As shown in
Fig. 3, treatment with etoposide and
mitomycin C resulted in an enhanced release of mono- and
oligonucleosomes into the cytoplasm in KBP over KBM cells. However,
exposure to 200 µM C2 ceramide or 40 ng/ml
TRAIL (Fig. 3) resulted in similar responses between PAF-R-negative and
-positive cells. These studies demonstrate that the presence of the
PAF-R results in enhanced apoptosis in response to chemotherapeutic
agents etoposide and mitomycin C.
To assess whether this proapoptotic effect of the PAF-R is a general
characteristic of GPCRs, KB cells were transduced with the fMLP-R. This
GPCR was chosen as a control since epithelial cells, unlike bacteria,
do not produce the peptide ligand fMLP. Calcium mobilization assays
with Indo-1-loaded KBF cells resulted in intracellular calcium
responses to 100 nM fMLP confirming that the fMLP-R was
functional (data not shown). Consistent with the notion that
chemotherapeutic agents are not activating GPCRs in a
ligand-independent fashion, transducing KB cells with the fMLP-R did
not affect responses to etoposide or mitomycin C (Fig.
4).
Effects of Ablation of the Epidermal PAF-R on Chemotherapeutic
Agent-induced Cytotoxicity--
The next studies were designed to
assess whether endogenous levels of the epidermal PAF-R could affect
responsiveness of the cells to chemotherapeutic agents. To address this
question HaCaT keratinocytes that express native PAF-Rs were transduced
with a retrovirus from which an antisense RNA corresponding to the PAF-R mRNA could be induced (HaCaTpRevTRE-as-PAFR). Radioligand binding studies with the PAF-R antagonist [3H]WEB 2086 were used to assess the ability of the antisense system to ablate PAF-R
expression in HaCaT cells. Binding studies using 10 nM
[3H]WEB 2086 ± 1 µM PAF revealed
56 ± 13 (mean ± S.E., n = 3) fmol of
[3H]WEB 2086 specifically bound/106 untreated
HaCaTpRevTRE-as-PAFR cells. In contrast, KBP cells specifically bound
357 ± 73 fmol of [3H]WEB 2086/106
cells, and KBM cells did not specifically bind [3H]WEB
2086. HaCaTpRevTRE-as-PAFR cells treated with 10 mg/ml doxycycline for
48 h did not exhibit specific [3H]WEB 2086 binding.
Doxycycline treatment of control HaCaTpRevTRE cells did not affect
[3H]WEB 2086-specific binding (48 ± 8 in untreated
versus 55 ± 9 fmol/106 cells in treated
cells). Measurements of intracellular calcium flux or IL-8 production
in response to PAF-R agonists were used as functional tests to confirm
ablation of endogenous PAF-R expression in doxycycline-treated
HaCaTpRevTRE-as-PAFR cells. As shown in Fig.
5A, doxycycline (10 mg/ml)
treatment of HaCaTpRevTRE-as-PAFR cells inhibited PAF-induced
intracellular calcium flux yet had no effect on calcium mobilization
induced by bradykinin, which acts via a separate GPCR. Similarly
doxycycline pretreatment prevented CPAF-induced but not phorbol
12-myristate 13-acetate-induced IL-8 production in HaCaTpRevTRE-as-PAFR
cells (Fig. 5B). Consistent with the radioligand binding
studies, doxycycline pretreatment did not affect Ca2+
mobilization responses or IL-8 production by PAF or the
non-metabolizable PAF-R agonist CPAF in control HaCaTpRevTRE cells
(Fig. 5, A and B). These studies confirm the
ability to ablate PAF-R expression in HaCaT cells using a novel
retroviral Tet-inducible system.
Ablation of the endogenous PAF-R in HaCaT cells resulted in a
diminishment of caspase-3 induction due to chemotherapeutic agents
mitomycin C and etoposide, yet it did not affect responses to
C2 ceramide and TRAIL (Fig. 5C). Similar
findings were obtained when apoptosis was measured by release of mono-
and oligonucleosomes by enzyme-linked immunosorbent assay (Fig.
5D). These studies indicate that endogenous levels of PAF-R
expression in carcinomas are adequate to modulate chemotherapeutic
agent-induced apoptosis.
The Role of Oxidative Stress on PAF-R Augmentation of
Chemotherapeutic Agent-induced Cytotoxicity--
The finding that the
epidermal PAF-R could augment apoptosis due to etoposide and mitomycin
C led to further studies defining whether this was occurring
secondarily to the pro-oxidative characteristics of these agents. To
test this, KB cells were loaded with the
H2O2-sensitive fluorescent dye CM-DCFDA,
and levels of intracellular H2O2 were measured.
As has been reported previously (26), activation of the epidermal PAF-R
with CPAF resulted in increased intracellular H2O2 (Fig. 6).
Consistent with their reported ability to induce a pro-oxidative stress
(27, 28), both etoposide and mitomycin C treatment resulted in
increased intracellular H2O2 levels in KB
cells. The levels of intracellular H2O2
generated in response to chemotherapeutic agents were greater in KBP
than in KBM cells, which is consistent with the hypothesis that
treatment of cells with etoposide or mitomycin C can activate the
PAF-R. Treatment with TRAIL (Fig. 6) or C2 ceramide (not
shown) did not significantly affect intracellular
H2O2 levels.
The next studies used antioxidants to confirm the involvement of
oxidative stress in the ability of chemotherapeutic agents to activate
the epidermal PAF-R and thus modulate apoptosis. Pretreatment of KB
cells with the antioxidants trolox and resveratrol (29) blunted
etoposide-mediated caspase-3 induction selectively in KBP cells (Fig.
7). These antioxidants similarly blocked
caspase-3 induction by mitomycin C by ~50% in KBP cells (not shown).
However, pretreatment with these two antioxidants did not affect
apoptosis induced by etoposide or mitomycin C in KBM cells nor did it
alter the effects of TRAIL or C2 ceramide in either KBM or
KBP cells (not shown). Altogether these studies provide solid
support for the hypothesis that oxidative stress induced by
chemotherapeutic agents results in PAF-R activation, which can augment
the proapoptotic effects of these agents.
Involvement of the NF-
To test whether PAF-R-mediated activation of the NF-
To assess whether PAF-R-mediated activation of the NF-
The presence of the super-repressor I These studies provide evidence that in epithelial carcinoma cells
the PAF-R can augment apoptosis induced by chemotherapeutic agents via
an NF- Recent studies have shown that oxidative stress can trigger the
production of lipids with PAF-R agonist activity. For example, chemical
oxidation of low density lipoproteins results in the production of a
PAF-R activity that has been shown to consist of fragmented alkyl GPCs
including 1-hexadecyl-2-butanoyl-GPC and 1-hexadecyl-2-butenoyl-GPC
along with trace levels of authentic PAF (15). Of significance,
systemic exposure to the strong oxidative stress of tobacco smoke has
been shown to induce the production of these PAF-R agonists in hamsters
in vivo (32). Other pro-oxidative stressors such as UVB
radiation can also stimulate the PAF-R through production of PAF
and PAF-like lipids (7, 13). Several lines of evidence suggest that
chemotherapeutic agents etoposide and mitomycin C are activating the
PAF-R through the production of PAF/PAF-like species. First, these
chemotherapeutic agents are known pro-oxidative stressors (27, 28);
this is confirmed in the present studies using the fluorescent dye
CM-DCFDA to measure intracellular H2O2
(Fig. 6). Other proapoptotic stimuli that do not induce an oxidative
stress (C2 ceramide and TRAIL) did not have differential
effects on KBM versus KBP cells. Second, pretreatment with
antioxidants blocked the augmentation of chemotherapy-mediated apoptosis found in PAF-R-expressing KBP yet did not affect that in KBM
cells. The finding that expression of the GPCR for fMLP in KB cells
(KBF) did not affect chemotherapy-induced apoptosis suggests that these
agents were not nonspecifically activating the GPCR in a
ligand-independent fashion. Thus, the data presented in these studies
support the hypothesis that these chemotherapeutic agents have the
ability to induce the production of PAF/PAF-like species. Ongoing
studies are attempting to define the structural identity of
chemotherapy-induced PAF-R agonistic activity.
The current studies also begin to define mechanistically how PAF-R
activation can augment apoptosis by chemotherapeutic agents. GPCRs
including the PAF-R have been shown to activate the NF- Although expression of the fMLP-R in KB cells did not affect
chemotherapy-induced apoptosis, this receptor was found to have in
common with the PAF-R the ability to activate the NF- The mechanism(s) by which the NF- Along with the effects of NF- In summary, the present studies demonstrate that the presence of the
epidermal PAF-R results in augmentation of the cytotoxic effects of
etoposide and mitomycin C. We describe a novel pathway by which these
pro-oxidative stressors induce PAF/PAF-like species that then activate
the NF-B (NF-
B) are found to be involved in this augmentative effect because it was blocked by
antioxidants and inhibition of the NF-
B pathway using a
super-repressor form of inhibitor B. These studies provide evidence for
a novel pathway whereby the epidermal PAF-R can augment
chemotherapy-induced apoptotic effects through an
NF-
B-dependent process.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
, and eicosanoids (12-14).
as well as the apoptotic
response of ultraviolet B radiation in carcinoma cell lines, processes
inhibited by antioxidants (7, 8, 13, 16).
B proteins, sequence-specific transcription factors induced in response to inflammatory and other stressful stimuli (for a review, see Ref. 17).
Of note, activation of NF-
B has been shown to protect epidermal
cells against the apoptotic effects of TRAIL and tumor necrosis
factor-
via enhanced expression of inhibitor of apoptosis proteins
(18, 19). The stimuli that can induce this antiapoptotic response
include IL-1
,
interferon, and PAF. Although associated with a
protective response in this setting, activation of the NF-
B pathway
also has been shown to augment proapoptotic responses (20-22). The
current findings describe a putative mechanism by which this G
protein-coupled receptor can augment pro-oxidative and proapoptotic
stressors in epithelial cells and could provide a novel pathway by
which carcinomas can be more susceptible to chemotherapy-induced cytotoxicity.
MATERIALS AND METHODS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
was purchased from Peprotek (Rocky
Hill, NJ).
B
(I
BM)--
The I
BM containing the S32A and S36A
mutation of I
B
has been described previously (23), and the
retroviral DNA vector MIEG3, which uses enhanced green fluorescent
protein as the selectable marker, was a kind gift of Dr. David Williams
(University of Cincinnati) (24). To create KBM and KBP cells stably
expressing I
BM, the I
BM cDNA was subcloned into the
EcoRI site of MIEG3, and orientation was assessed by
restriction endonuclease mapping and sequencing. Infectious amphotropic
retroviruses were produced from both MIEG-I
BM and control MIEG
backbone by transient transfection using standard protocols (19).
Briefly, the Phoenix amphotropic packaging cell line was transfected
with the DNA constructs using FuGENE 6, and the supernatants collected
48 h later containing infectious virions were then used to infect
KBM/KBP cells. Transduced cells were sorted by a fluorescence-activated
cell sorter on the basis of enhanced green fluorescent protein expression.
B Reporter Assay--
Cells were plated at a density of
1.5 × 106 cells in a 10-cm dish and allowed to
stabilize for 1 day. Cells were then transfected using FuGENE 6 (Roche
Applied Science) with 10 µg of NF-
B-luciferase reporter plasmid
(pNF-
B-luc) and 10 µg of pCMV-
-galactosidase as an internal
control for the transfection efficiency. 24 h after transfection
cells were treated with CPAF, IL-1
, or chemotherapeutic agents or
were mock-treated and then harvested in reporter lysis buffer (Promega)
following an additional 6-h incubation. 20-µl aliquots of the lysates
were assayed for
-galactosidase and luciferase activities using an
LB9501 luminometer (Lumat). Luciferase activities were normalized for
each transfection using the control
-galactosidase activities.
RESULTS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
View larger version (28K):
[in a new window]
Fig. 1.
Dose-dependent effects of
etoposide, mitomycin C, C2 ceramide, and TRAIL on caspase-3
enzyme activation in KBM versus KBP cells. KB
cells were treated with various doses of etoposide (A),
mitomycin C (B), C2 ceramide (C), or
TRAIL (D) for 16 h. Following the treatment duration,
the cells were collected and lysed, and caspase-3-specific activity was
determined as an index of apoptotic activity after treatment. The
values are mean ± S.D. of duplicate samples from a typical
experiment from four to six separate experiments. CON,
control.
View larger version (31K):
[in a new window]
Fig. 2.
Time-dependent effects of
etoposide, mitomycin C, C2 ceramide, and TRAIL on caspase-3
enzyme activation in KBM versus KBP cells. KB
cells were treated with 6 µg/ml etoposide (A), 5 µg/ml
mitomycin C (B), 200 µM C2
ceramide (C), or 40 ng/ml TRAIL (D) for various
times ranging from 0 to 24 h. Following the treatment duration,
the cells were collected and lysed, and caspase-3-specific activity was
determined as an index of apoptotic activity after treatment. The
values are mean ± S.D. of duplicate samples from a typical
experiment from three to four separate experiments. CON,
control.
View larger version (27K):
[in a new window]
Fig. 3.
Effects of etoposide, mitomycin
C, C2 ceramide, and TRAIL on nucleosome enrichment in KBM
versus KBP cells. KB cells were treated with 6 µg/ml etoposide, 5 µg/ml mitomycin C, 200 µM
C2 ceramide, or 40 ng/ml TRAIL for 16 h. Following the
treatment duration, the cells were collected and lysed, and enrichment
of nucleosomes into the cytoplasm was determined as an index of
apoptotic activity in KBP cells. Data represent the -fold difference in
activity or enrichment compared with untreated control
(CON). The values are the mean ± S.D. of enhancement
from a typical experiment from three to five separate experiments.
ETOP, etoposide; MMC, mitomycin C;
CER, C2 ceramide.
View larger version (30K):
[in a new window]
Fig. 4.
Effects of etoposide, mitomycin C,
C2 ceramide, and TRAIL on caspase-3 enzyme activation in
KBP versus KBF cells. KB cells were
treated with 6 µg/ml etoposide, 5 µg/ml mitomycin C, 200 µM C2 ceramide, or 40 ng/ml TRAIL for 16 h. Following the treatment duration, the cells were collected and
lysed, and caspase-3-specific activity was determined as an index of
apoptotic activity after treatment. The values are mean ± S.D. of
a typical experiment from four separate experiments. ETOP,
etoposide; MMC, mitomycin C; CER, C2
ceramide; CON, control.
View larger version (23K):
[in a new window]
Fig. 5.
Effect of ablation of the endogenous PAF-R on
caspase-3 enzyme activation in HaCaT cells. HaCaTpRevTRE and
HaCaTpRevTRE-as-PAFR cells were treated with 10 mg/ml doxycy- cline for 48 h to allow induction of as-PAFR construct.
Following induction, HaCaT cells were tested as follows. A,
intracellular calcium mobilization responses were assessed in
Indo-1/AM-loaded HaCaT cells in response to 100 nM CPAF or
100 nM bradykinin. B, IL-8 release into
supernatants was assessed by enzyme-linked immunosorbent assay
following an 8-h incubation of 100 nM CPAF or 1 µM phorbol 12-myristate 13-acetate (PMA).
Caspase-3 enzyme levels (C) or enrichment of nucleosomes
into the cytoplasm (D) was determined as an index of
apoptotic activity assessed 16 h after treatment with 6 µg/ml
etoposide, 5 µg/ml mitomycin C, or 40 ng/ml TRAIL. * denotes
statistically significant (p < 0.05) differences
between HaCaTpRevTRE and HaCaTpRevTRE-as-PAFR cells. BK,
bradykinin; ETOP, etoposide; MMC, mitomycin C;
CER, C2 ceramide; CON, control.
View larger version (28K):
[in a new window]
Fig. 6.
Effect of chemotherapeutic agents on
intracellular H2O2 levels in KBM
versus KBP cells. KB cells were loaded with a 10 µM concentration of
H2O2-sensitive fluorescent dye CM-DCFDA,
and intracellular H2O2 levels were assessed
after 6 h in response to 100 nM CPAF, 6 µg/ml
etoposide, 5 µg/ml mitomycin C, 40 ng/ml TRAIL, or 0.6%
H2O2. ETOP, etoposide;
MMC, mitomycin C; CER, C2 ceramide;
CON, control.
View larger version (31K):
[in a new window]
Fig. 7.
Effect of antioxidants on etoposide-induced
caspase-3 enzyme activation in KB cells. KB cells were pretreated
for 30 min with a 10 mM concentration of antioxidants
trolox and resveratrol or Me2SO vehicle before
addition of 6 µg/ml etoposide. Following a further 16-h treatment
duration, the cells were collected and lysed, and caspase-3-specific
activity was determined as an index of apoptosis. The values are
mean ± S.D. of a typical experiment from three separate
experiments. ETOP, etoposide; TRO, trolox;
RES, resveratrol; CON, control.
B System in PAF-R-mediated Augmentation of
Chemotherapeutic Agent-induced Cytotoxicity--
The mechanism by
which the PAF-R could augment the cytotoxic effects of chemotherapeutic
agents is not known. Indeed activation of the epidermal PAF-R is linked
to numerous signal transduction pathways that could be responsible for
the augmented apoptotic effects (10). It should be noted that like a
diverse group of stimuli including pro-oxidative stressors the
epidermal PAF-R induces the transcription factor NF-
B, and
activation of this same pathway has also been reported to have potent
proapoptotic effects in various cell types (20-22).
B pathway was
involved in the augmentation of chemotherapy-mediated apoptosis, we
examined the ability of etoposide and mitomycin C to activate the
NF-
B system in KB cells as well as whether a dominant-negative inhibitor of NF-
B could affect PAF-R-mediated augmentation of apoptosis in response to these chemotherapeutic agents. First, gel
shift studies were used to examine levels of NF-
B in
chemotherapeutic agent-treated KBP and KBF cells (Fig.
8A). These studies
demonstrated increased NF-
B binding in KBP cells in response to
treatment with the PAF-R agonist CPAF. KBF cells responded to fMLP in
similar fashion. However, treatment of KB cells with etoposide or
mitomycin C resulted in an enhanced level of NF-
B binding activity
in KBP over KBF cells. Transfection of KB cells with an
NF-
B-luciferase reporter plasmid to measure NF-
B activity also
revealed increased levels in KBP cells over KBM cells in response to
treatment with etoposide and mitomycin C (Fig. 8B). These
data indicate that these chemotherapeutic agents are functionally
coupled to a biochemical pathway that results in NF-
B activation
through the PAF-R.
View larger version (62K):
[in a new window]
Fig. 8.
Effect of chemotherapeutic agents on
NF- B binding and activation in KB
cells. A, following a 1-h treatment with 100 nM
CPAF, 100 nM fMLP, 6 µg/ml etoposide, 5 µg/ml mitomycin
C, or 25 ng/ml IL-1
, KB cells were harvested and lysed. For NF-
B
binding whole cell extracts were obtained through three cycles of
freeze-thaw and diluted to a concentration of 2 µg/ml in lysis
buffer. Whole cell extracts were incubated with radiolabeled NF-
B
probe or OCT-1 control and electrophoretically separated.
B, KBM or KBP cells transfected with NF-
B luciferase
reporter gene were stimulated with 6 µg/ml etoposide, 5 mg/ml
mitomycin C, 100 nM CPAF, or 25 ng/ml IL-1
for 6 h,
and relative luciferase activity was normalized to
-galactosidase. *
denotes statistically significant differences between KBP and KBM
cells. ETOP, etoposide; MMC, mitomycin C;
CON, control; no txt, no
treatment.
B pathway was
exerting a protective or augmentative effect on chemotherapy-induced apoptosis, KBM and KBP cells were transfected with a super-repressor I
B protein (KBM-MIEG-I
BM and KBP-MIEG-I
BM). We have previously shown that increased I
B
degradation and increased NF-
B binding activity induced by PAF-R agonist CPAF and IL-1
were blocked in
these cells transduced with this mutant non-degradable I
B protein
(19).
B protein did not affect the
cytotoxic effects of etoposide or mitomycin C in PAF-R-negative KBM
cells (Fig. 9). However, blockade of the
NF-
B pathway inhibited the PAF-R-induced augmentation of
apoptosis seen with this pro-oxidative chemotherapeutic agent.
Levels of apoptosis induced by C2 ceramide (Fig. 9) or
TRAIL (19) were similar in all KB cell types. Survival studies using
trypan blue dye exclusion as a measure of cell viability at 24 and
48 h following treatment with etoposide and mitomycin C mirrored
the caspase-3 studies demonstrating increased survival in
KBP-I
BM over KBP-MIEG cells (not shown). These studies
indicate that activation of the NF-
B pathway is responsible for
PAF-R augmentation of chemotherapeutic agent-induced cytotoxicity.
View larger version (29K):
[in a new window]
Fig. 9.
Effect of inhibition of the
NF- B pathway on PAF-R augmentation of
chemotherapeutic agent-induced apoptosis in KB cells. KBM and KBP
cells transduced with I
BM or MIEG vector control retrovirus were
treated with 6 µg/ml etoposide, 5 µg/ml mitomycin C, or 200 µM C2 ceramide, and caspase-3-specific
activity was determined 16 h later. The values are mean ± S.D. of a typical experiment from four separate experiments.
ETOP, etoposide; MMC, mitomycin C;
CER, C2 ceramide; CON, control.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
B-dependent process. The augmentation of
chemotherapeutic agent-induced apoptosis was mediated through the PAF-R
as it was only seen in PAF-R-expressing KBP and HaCaT carcinoma cells,
and ablation of the endogenous PAF-R with an inducible antisense
strategy blocked this increased cytotoxic effect. Our results describe a novel function for a G protein-coupled receptor, namely the induction
of an NF-
B-dependent proapoptotic pathway by which the
PAF-R can augment the effectiveness of chemotherapeutic agents.
B system
(19). Consistent with the involvement of NF-
B in the augmentation of
chemotherapy-induced apoptosis, etoposide and mitomycin C treatment of
KBP cells resulted in a much greater level of NF-
B binding activity
than that in KBM cells. Blocking NF-
B activation with a
super-repressor I
B mutant decreased the augmentative effect of PAF-R
expression on chemotherapy-induced apoptosis. Of note, preliminary
studies in our laboratory indicate that the augmentation of UVB
radiation-induced apoptosis in KBP cells is also blocked by ablation of
NF-
B activation (data not shown).
B pathway. Indeed fMLP treatment of KBF cells resulted in NF-
B binding by gel
shift assays (Fig. 8B) and induced I
B
degradation
(data not shown). Transfection of KBF cells with an NF-
B-luciferase reporter plasmid to measure NF-
B activity also revealed increased levels in KBF cells in response to fMLP. In fact, the effects of 100 nM fMLP on KBF cells were similar to those induced by 100 nM CPAF in KBP cells (data not shown). The ability of the
fMLP-R to stimulate the NF-
B system yet not to affect
chemotherapy-induced responses of NF-
B activation or apoptosis
supports the contention that these pro-oxidative chemotherapeutic
agents are activating the epidermal PAF-R via production of endogenous ligands.
B pathway can potentially promote
or protect against apoptosis in epithelial carcinomas is unclear
at this time and is an active area of study. Although NF-
B
activation by the PAF-R appears to be responsible for promoting apoptosis in response to chemotherapeutic agents or UVB radiation, we
have previously demonstrated that PAF-R activation of NF-
B protects
epithelial cells against apoptosis induced by TRAIL and tumor necrosis
factor-
(19). That PAF-R-mediated activation of the NF-
B system
could yield such dissimilar outcomes as either promoting or protecting
against proapoptotic stimuli is consistent with data from several
recent studies from a variety of cell types (18-22). For example, in
rat primary forebrain cultures
N-methyl-D-aspartate-induced apoptosis has been
shown to be dependent upon superoxide-mediated NF-
B activation (20).
Similar findings have been reported in glutamate-induced cytotoxicity
(21). One possible mechanism for NF-
B-mediated cytotoxicity could
involve its ability to up-regulate expression of p53 and Bax (20).
Ongoing studies are evaluating the effects of NF-
B activation on the
ratio of pro- and antiapoptotic protein expression in epithelial cells.
B translocation on apoptotic defenses,
another possible explanation for the differences in PAF-R-mediated NF-
B-dependent protection against or augmentation
of apoptosis lies in the nature of the proapoptotic stimuli.
Chemotherapeutic agents and UVB radiation are potent DNA-damaging
agents, and thus concomitant activation of the NF-
B system in the
presence of significant DNA damage might not allow antiapoptotic
proteins such as inhibitor of apoptosis proteins to be produced. Thus, other potential NF-
B-linked proapoptotic pathways could predominate.
B pathway through the PAF-R. The biological significance of
this pathway is not clear, but it could be the impetus for further
studies to define whether the presence of the PAF-R could modulate
chemotherapeutic responses in vivo. Finding that this novel
pathway is an important determinant in the effectiveness of certain
chemotherapeutic agents would be useful for planning chemotherapeutic
strategies in PAF-R-expressing tumor cells.
![]() |
ACKNOWLEDGEMENT |
---|
We thank Dr. Raymond Konger for critical reading of this manuscript.
![]() |
FOOTNOTES |
---|
* This research was funded in part by grants from the Showalter Memorial Foundation and the Riley Memorial Association and by National Institutes of Health Grants AR01993 and HL62996.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
¶ Supported by a grant from the Dermatology Foundation.
To whom correspondence should be addressed: H. B. Wells
Center for Pediatric Research, James Whitcomb Riley Hospital for
Children, Rm. 2659, Indiana University School of Medicine, 702 Barnhill Dr., Indianapolis, IN 46202. Tel.: 371-274-7705; Fax: 317-274-5378; E-mail: jtravers@iupui.edu.
Published, JBC Papers in Press, February 24, 2003, DOI 10.1074/jbc.M211287200
![]() |
ABBREVIATIONS |
---|
The abbreviations used are:
PAF, platelet-activating factor;
PAF-R, PAF receptor;
CPAF, 1-hexadecyl-2-N-methylcarbamoyl-glycerophosphocholine;
IL, interleukin;
GPC, glycerophosphocholine;
GPCR, G protein-coupled
receptor;
fMLP, N-formyl-methionyl-leucyl-phenylalanine;
fMLP-R, fMLP receptor;
TRAIL, tumor necrosis factor-related
apoptosis-inducing ligand;
NF-B, nuclear factor
B;
as-PAFR, antisense PAF-R;
Tet, tetracycline;
AMC, 7-amino-4-methylcoumarin;
CM-DCFDA, 5,6-chloromethyl-2',7'-dichlorodihydrofluorescein diacetate..
![]() |
REFERENCES |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
1. | Cohen, G. M. (1997) Biochem. J. 326, 1-16[Medline] [Order article via Infotrieve] |
2. | Jacobson, M. D., Weil, M., and Raff, M. C. (1997) Cell 88, 347-354[Medline] [Order article via Infotrieve] |
3. |
Reed, J. C.
(2000)
Am. J. Pathol.
157,
1415-1430 |
4. | Prescott, S. M., Zimmerman, G. A., Stafforini, D. M., and McIntyre, T. M. (2000) Annu. Rev. Biochem. 69, 419-445[CrossRef][Medline] [Order article via Infotrieve] |
5. | Michel, L., Denizot, Y., Thomas, Y., Jean-Louis, F., Heslan, M., Benveniste, J., and Dubertret, L. (1990) J. Investig. Dermatol. 95, 576-582[Abstract] |
6. | Travers, J. B., Harrison, K. A., Johnson, C. A., Clay, K. L., Morelli, J. G., and Murphy, R. C. (1996) J. Investig. Dermatol. 107, 88-94[Abstract] |
7. |
Barber, L. A.,
Spandau, D. F.,
Rathman, S. C.,
Murphy, R. C.,
Johnson, C. A.,
Kelley, S. W.,
Hurwitz, S. A.,
and Travers, J. B.
(1998)
J. Biol. Chem.
273,
18891-18897 |
8. |
Travers, J. B.
(1999)
J. Investig. Dermatol.
112,
279-283 |
9. | Alappatt, C., Johnson, C. A., Clay, K. L., and Travers, J. B. (2000) Arch. Dermatol. Res. 292, 256-259[CrossRef][Medline] [Order article via Infotrieve] |
10. | Ishii, S., and Shimizu, T. (2000) Prog. Lipid Res. 39, 41-82[CrossRef][Medline] [Order article via Infotrieve] |
11. | Travers, J. B., Huff, J. C., Rola-Plaeszczynski, M., Gelfand, E. W., Morelli, J. G., and Murphy, R. C. (1995) J. Investig. Dermatol. 105, 816-823[Abstract] |
12. |
Pei, Y.,
Barber, L. A.,
Murphy, R. C.,
Johnson, C. A.,
Kelley, S. A.,
Dy, L. C.,
Fertel, R. H.,
Nguyen, T. M.,
Williams, D. A.,
and Travers, J. B.
(1998)
J. Immunol.
161,
1954-1961 |
13. |
Dy, L. C.,
Pei, Y.,
and Travers, J. B.
(1999)
J. Biol. Chem.
274,
26917-26922 |
14. |
Walterscheid, J. P.,
Ullrich, S. E.,
and Nghiem, D. X.
(2002)
J. Exp. Med.
195,
171-179 |
15. |
Marathe, G. K.,
Davies, S. S.,
Harrison, K. A.,
Silva, A. R.,
Murphy, R. C.,
Castro-Faria-Neto, H.,
Prescott, S. M.,
Zimmerman, G. A.,
and McIntyre, T. M.
(1999)
J. Biol. Chem.
274,
28395-28404 |
16. | Countryman, N. B., Pei, Y., Yi, Q., Spandau, D. F., and Travers, J. B. (2000) J. Investig. Dermatol. 115, 267-272[CrossRef][Medline] [Order article via Infotrieve] |
17. | Foo, S. Y., and Nolan, G. P. (1999) Trends Genet. 15, 229-235[CrossRef][Medline] [Order article via Infotrieve] |
18. |
Kothny-Wilkes, G.,
Kulms, D.,
Poppelmann, B.,
Luger, T. A.,
Kubin, M.,
and Schwarz, T.
(1998)
J. Biol. Chem.
273,
29247-29253 |
19. |
Southall, M. D.,
Isenberg, J. S.,
Nakshatri, H.,
Yi, Q.,
Pei, Y.,
Spandau, D. F.,
and Travers, J. B.
(2001)
J. Biol. Chem.
276,
45548-45554 |
20. |
McInnis, J.,
Wang, C.,
Anastasio, N.,
Hultman, M.,
Ye, Y.,
Salvemini, D.,
and Johnson, K. M.
(2002)
J. Pharmacol. Exp. Ther.
301,
478-487 |
21. | Wang, Y., Qin, Z. H., Nakai, M., Chen, R. W., Chuang, D. M., and Chase, T. N. (1999) Neuroscience 94, 1153-1162[CrossRef][Medline] [Order article via Infotrieve] |
22. | Nakai, M., Qin, Z. H., Chen, J. F., Wang, Y., and Chase, T. N. (2000) J. Neurochem. 74, 647-658[CrossRef][Medline] [Order article via Infotrieve] |
23. |
Newton, T. R.,
Patel, N. M.,
Bhat-Nakshatri, P.,
Stauss, C. R.,
Goulet, R. J., Jr.,
and Nakshatri, H.
(1999)
J. Biol. Chem.
274,
18827-18835 |
24. |
Williams, D. A.,
Tao, W.,
Yang, F.,
Kim, C.,
Gu, Y.,
Mansfield, P.,
Levine, J. E.,
Petryniak, B.,
Derrow, C. W.,
Harris, C.,
Jia, B.,
Zheng, Y.,
Ambruso, D. R.,
Lowe, J. B.,
Atkinson, S. J.,
Dinauer, M. C.,
and Boxer, L.
(2000)
Blood
96,
1646-1654 |
25. | Kuhn, C., Hurwitz, S. A., Kumar, M. G., Cotton, J., and Spandau, D. F. (1999) Int. J. Cancer 80, 431-438[CrossRef][Medline] [Order article via Infotrieve] |
26. | Goldman, R., Moshonov, S., and Zor, U. (1999) Biochim. Biophys. Acta 1438, 349-358[Medline] [Order article via Infotrieve] |
27. |
Kagan, V. E.,
Kuzmenko, A. I.,
Tyurina, Y. Y.,
Shvedona, A. A.,
Matsura, T.,
and Yalowich, J. C.
(2001)
Cancer Res.
61,
7777-7784 |
28. | Xu, B. H., and Singh, S. V. (1992) Cancer Lett. 66, 49-53[Medline] [Order article via Infotrieve] |
29. |
MacCarrone, M.
(1999)
Eur. J. Biochem.
265,
27-34 |
30. | Deleted in proof |
31. | Deleted in proof |
32. |
Lehr, H. A.,
Weyrich, A. S.,
Saetzler, R. K.,
Jurek, A.,
Arfors, K. E.,
Zimmerman, G. A.,
Prescott, S. M.,
and McIntyre, T. M.
(1997)
J. Clin. Investig.
99,
2358-2364 |