From the Institute of Chemical Toxicology, Wayne State University, Detroit, Michigan 48201
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ABSTRACT |
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The relationship between aryl hydrocarbon
receptor (AHR) content and susceptibility to apoptosis was examined in
the murine hepatoma 1c1c7 cell line and a series of variants having
different levels of AHR expression. Exposure of 1c1c7 cultures to
N-acetylsphingosine (C2-ceramide) caused a
concentration-dependent inhibition of cell proliferation,
loss of viability, and induction of apoptosis as monitored by analyses
of DNA fragmentation and caspase activation. A variant cell line (Tao)
having ~10% of the AHR content of 1c1c7 cells also arrested
following exposure to C2-ceramide, but did not undergo
apoptosis. Modulation of 1c1c7 and Tao AHR contents by transfection of
Ahr antisense and sense constructs, respectively, confirmed
the relationship between AHR content and susceptibility to
C2-ceramide-induced apoptosis. C2-ceramide also
induced the apoptosis of an AHR-containing cell line lacking the aryl
hydrocarbon receptor nuclear translocator protein. AHR ligands
(i.e. 2,3,7,8-tetrachlorodibenzo-p-dioxin and
The aryl hydrocarbon receptor
(AHR)1 is a ligand-activated
transcription factor (1-3). Numerous xenobiotics, including
2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), are ligands of
the AHR. In many tissues and cell lines, the non-liganded AHR appears
to be a cytosolic protein (4, 5). Upon binding TCDD, the AHR
translocates to the nucleus where it forms a dimer with the aryl
hydrocarbon receptor nuclear translocator (ARNT) protein. AHR/ARNT
heterodimers interact with specific enhancer sequences in target genes
designated dioxin-responsive elements, and stimulate the
transcriptional activation of such genes (1-3). Many of the biological
processes initiated by TCDD can be attributed to this transcriptional
activation of target genes (1).
In general, the cellular functions of the AHR have been defined or
inferred from comparisons of the effects of ligands of the AHR in wild
type and AHR-deficient or null cell lines or mice (1-3,6-11). Such
approaches have delineated the need for the AHR in mediating the
teratogenic, immunomodulating, cytostatic, and apoptotic activities of
TCDD, and the role of the AHR in TCDD-initiated, transcriptional
activation of several phase I and II genes involved in
biotransformation (1-3, 6-11). However, studies employing exogenous
ligands do not address the issue of whether the AHR, in the absence of
exogenous ligand, has other cellular functions. Recent studies,
performed in the absence of exogenous ligand, have implicated a role
for the AHR in cell cycle progression. These studies used murine
hepatoma 1c1c7 and Tao cells, the latter being an AHR-deficient variant
derived from the 1c1c7 line (12). The doubling time of Tao cells is
considerably longer than parental 1c1c7 cells due to a prolongation of
G1, but can be shortened by the introduction of an
Ahr expression construct (12). Conversely, introduction of
an Ahr antisense expression construct into 1c1c7 cells
increases cell doubling times due to a lengthening of G1. This inverse relationship between AHR content and cell doubling times
has also been observed with rat hepatoma 5L and BP8 cells, the latter
being an AHR-deficient variant derived from 5L cells (11). An
unresolved issue in these studies is whether the observed AHR-dependent effects were ARNT-dependent and
mediated by an endogenous AHR ligand.
Apoptosis is a physiological process that entails the programmed death
of a cell. It plays a critical role during development and in the
maintenance of tissue and organ homeostasis (13, 14). Indeed,
disregulation of processes controlling apoptosis often contribute to
the development of neoplasia. Apoptosis is also the process by which
many genotoxic and chemotherapeutic drugs exert their cytotoxic
effects. Several studies implicate the AHR as having a role in
influencing or mediating apoptotic processes. For example,
immunohistochemical analyses of embryonic tissues show that AHR
expression is developmentally regulated and occurs independently of
ARNT expression in regions undergoing remodeling, a process that
involves apoptosis (15, 16). Similarly, resting T cells express very
low levels of the AHR (17, 18). Upon mitogenic stimulation they enter
the cell cycle, divide, and subsequently undergo apoptosis (19, 20).
Expression of the AHR is markedly increased following such stimulation,
and the kinetics of expression parallel the onset of apoptosis (17, 18). Although the role of the AHR in the above examples is
speculative, there are at least two examples in which the AHR protein
has been clearly associated with an apoptotic process. The first of
these entails the thymic atrophy seen in AHR+/+ mice, but
not AHR null mice, following exposure to TCDD (9, 21). The second
example relates to the fibrosis and reduced liver size seen in AHR null
mice (6, 7). Recent studies suggest that these phenotypic traits
reflect the autocrine production by AHR null hepatocytes of a cytokine
(e.g. transforming growth factor- Ceramide is an endogenous lipid generated by either de novo
biosynthesis or sphingomyelinase degradation of sphingomyelin (23, 24).
Intracellular levels of ceramide are elevated by a variety of
stimuli/agents that induce apoptosis, including Fas ligand engagement
of CD95, ionizing radiation, ultraviolet radiation, chemotherapeutic
and genotoxic chemicals, and several cytokines (23-26). Numerous
physiological processes are affected by ceramide. It has been reported
to be a modulator of immune cell differentiation, mitochondrial
respiration, inflammation, cell cycle progression, apoptosis, and the
stress response (23, 24, 27-29). Many of these processes are also
affected by TCDD and other ligands of the AHR. Furthermore, ceramide
has several structural features which suggest that it might be an AHR
ligand. Specifically, structure-activity analyses suggest that the AHR
preferentially binds aromatic, hydrophobic planar molecules, and has a
binding site with dimensions of ~6.8 × 13.7 Å (30, 31).
Although not aromatic, ceramide is a hydrophobic molecule. The amide
group, which serves as the link between ceramide's two acyl chains,
imparts a planar conformation on the molecule in which the axes of the
two acyl chains lie parallel to one another (32). Furthermore, the
overall dimensions of some ceramide molecules do not dramatically
differ from those of some AHR ligands (e.g. 4 × 20 Å for palmitoyl ceramide (C16); Ref. 33).
The current studies evolved from a preliminary characterization of
ceramide as a putative AHR ligand. As an approach we examined the
transcriptional activation of Cyp1a1 in AHR-containing
(1c1c7) and AHR-deficient (Tao) cells treated with
N-acetylsphingosine, a cell-permeable analog of ceramide.
Although initial studies did not support the hypothesis being tested,
we noted a pronounced difference in the viability of the two cell lines
following ceramide exposure.2
In the current investigation, we determined whether manipulation of the
AHR contents of cells of the 1c1c7 lineage could modulate susceptible
to ceramide-induced apoptosis, and two additional inducers of
apoptosis: staurosporine and doxorubicin (23, 34). Susceptibility of
the various 1c1c7 variant lines to ceramide-induced apoptosis
correlated directly with cellular AHR contents, but was independent of
the presence of a functional ARNT protein. In contrast, AHR content did
not influence susceptibility to staurosporine- or doxorubicin-mediated
apoptosis. These findings implicate a novel role for AHR in
ceramide-mediated apoptosis that is independent of its functioning as
an ARNT-associated transcription factor.
Materials--
Cell Culture--
The murine hepatoma 1c1c7, BPrc1,
Tao, WCMV, WARV, TCMV, and TAHR cell lines were obtained from Dr. J. Whitlock, Jr. (Stanford University, Palo Alto, CA). The origins and
characterizations of these cell lines have been described in detail
(12, 35, 36). All cell lines were cultured at 32 °C in
Analyses of a chemical's cytostatic/cytotoxic effects were performed
with cultures initially seeded at a density of 5-20 × 104 cells/60-mm dish in 3 ml of medium. The following day
cultures were treated with either solvent (Me2SO or
ethanol), ceramides (dissolved in Me2SO or ethanol), or
TCDD, Caspase Assay--
Cells were plated at a density of ~1 × 106/100-mm culture dish in 10 ml of medium. Two days
after plating, cultures were treated with either solvent or chemicals
of interest. At various times after treatment, cells were dislodged
into the culture medium with a cell scraper, pelleted by
centrifugation, resuspended in 0.5 ml of lysis buffer (10 mM Tris, pH 7.5, 130 mM NaCl, 1% Triton X-100,
10 mM NaF, 10 mM NaPi, and 10 mM NaPPi), quick frozen in liquid nitrogen, and
stored at DNA Fragmentation Assay--
Cells were plated at ~1.5 to
2 × 106/100-mm dish in 10 ml of medium. Cultures were
treated 1 day later with solvent or chemicals. At various times after
treatment, the culture medium was removed and the cultures were washed
with phosphate-buffered saline, aspirated dry, and stored at
Adherent cells were lysed in the plates by addition of 5 ml of 4 M guanidine isothiocyanate, 25 mM sodium
citrate, pH 7.0, 100 mM RNA Preparation and Northern Blot Analyses--
Total cellular
RNA was isolated according to the acidic phenol extraction method of
Chomczynski and Sacchi (37). RNA was resolved on 1.2%
agarose/formaldehyde gels and transferred to nylon membranes as
described previously (5). The probes used for the detection of CYP1A1
and 7 S RNAs, and the conditions used for hybridization have been
described in detail (38).
Immunoblotting and Quantification of AHR--
The conditions
detailed by Reiners et al. (5) were used for the preparation
of cellular extracts, separation of proteins on SDS-polyacrylamide
gels, transfer of proteins to nitrocellulose, and immunodetection of
the AHR with an affinity-purified polyclonal rabbit antibody to the
murine AHR protein (provided by Dr. A. Poland while at the University
of Wisconsin, Madison, WI). Antigen-immunoglobulin conjugates were
detected with an ECL detection kit (Amersham Pharmacia Biotech) and
recorded on x-ray film.
Ceramide Induction of Apoptosis in 1c1c7 and AHR-deficient Variant
Cell Lines--
A variety of cell types commit to, and undergo
apoptosis upon exposure to N-acetylsphingosine
(C2-ceramide), a cell-permeable analog of ceramide (23,
39). Exposure of 1c1c7 cultures to varying concentrations of
C2-ceramide resulted in a
concentration-dependent suppression of cell proliferation
(Fig. 1A). Concentrations
Tao cells were derived from 1c1c7 cells and have been reported to
contain normal amounts of ARNT protein, but only 10% of the AHR
protein content of 1c1c7 cells (12, 36). The data present in the
top panel of Fig. 2
confirm the markedly different AHR contents of the 1c1c7 and Tao cell
lines. These differences were also reflected in how the two cell lines
responded to TCDD. Although steady state CYP1A1 mRNA levels were
elevated above basal levels in both cell lines following TCDD exposure,
the induced CYP1A1 mRNA contents of 1c1c7 cells were severalfold
greater than those observed in its AHR-deficient counterpart
(bottom panel of Fig. 2).
Tao cells grew considerably slower than 1c1c7 cells (Fig. 1, compare
A and B). Proliferation of Tao cells, like the
parental 1c1c7 line, was completely suppressed by exposure to
concentrations of C2-ceramide
Ma and Whitlock (12) recently reported the characterization of a series
of cell lines derived by transfection of 1c1c7 and Tao cells with
Ahr antisense and sense expression constructs, respectively.
Transfection of 1c1c7 cells with an Ahr antisense construct
gave rise to a cell line designated WARV having AHR contents similar to
those measured in Tao cells (top panel of Fig.
2). Conversely, transfection of Tao cells (also called AhR-D; Ref. 12)
with an Ahr sense expression vector gave rise to a cell line
designated TAHR having AHR contents similar to that seen in 1c1c7 cells
(Fig. 2). The AHR contents of 1c1c7 and Tao cells stably transfected
with just the vector (resulting lines are designated WCMV and TCMV,
respectively) were very similar to the contents of the corresponding
parental cells (Fig. 2). The responsiveness of the four engineered cell
lines to TCDD, as monitored by measuring steady state CYP1A1 mRNA
contents 6 h after treatment, also correlated with cellular AHR
contents (bottom panel of Fig. 2). We used these
lines to further examine the relationship between AHR content and
susceptibility to ceramide-induced apoptosis (Fig.
3). The sensitivity of WCMV cells to
C2-ceramide was virtually identical to the parental line
(compare Fig. 1, A and C, with Fig. 3,
A and E). However, WARV cells were markedly less
sensitive to C2-dependent killing, as judged by
trypan blue staining (Fig. 3, compare E and F)
and DNA laddering (Fig. 3, I and J). Conversely,
TAHR cells were more susceptible to C2-ceramide-induced killing and DNA fragmentation than the TCMV cell line (Fig. 3, compare
G with H, and K with
L).
The data presented in Fig. 3 merit two additional comments. First,
manipulation of AHR contents modulated cell doubling times. AHR-deficient variant lines (WARV and TCMV) grew much slower than the
engineered cell lines (WCMV and TAHR) expressing wild type AHR levels
(Fig. 3, compare A, B, C, and
D). These results are similar to those reported by Ma and
Whitlock (12). Second, irrespective of AHR content, proliferation was
arrested in all four engineered cell lines following exposure to 30 µM C2-ceramide (Fig. 3, A-D).
The studies reported in Figs. 1 and 3 were performed with cultures
grown at 32 °C in the presence of 5% fetal bovine serum (our
standard conditions; Ref. 38). Similar studies were performed with
cultures grown in variable amounts of FBS, or at 37 °C.2
The concentration-response curves were influenced by the amount of
serum present in the culture medium. Decreasing media serum concentrations decreased the amount of ceramide required to achieve cell killing. Increasing the culturing temperature to 37 °C slightly accelerated the kinetics of cell killing. However, the differential responsiveness of the parental and variant cell lines to
C2-ceramide was unaffected by either temperature or serum
content.2 Furthermore, the differential responsiveness of
the 1c1c7 and Tao lines was also observed when cultures were treated
with N-hexanoylsphingosine (C6-ceramide),
another cell-permeable analog of ceramide having a longer acyl
chain.2
Detection of Caspase Activity--
Endonuclease activation and
fragmentation of DNA into nucleosome multimers occurs in the
later stages of the apoptotic process. Activation of a series of
cysteine proteases, referred to as caspases, occurs earlier. Caspases 3 and 7 are activated by a variety of apoptotic inducers, and are
routinely assayed by monitoring cleavage of the synthetic substrate
N-acetyl-Asp-Glu-Val-Asp-7-amino-4-methylcoumarin (Ac-DEVD-AMC; Refs. 40 and 41). Activated caspases 3 and 7 cleave
Ac-DEVD-AMC between Asp and AMC, resulting in the release of a
fluorescent product.
Caspase specific activities in 1c1c7 (Fig.
4A) or TAHR cells (Fig.
4B) increased ~10-20-fold over 72 h following
exposure to the solvent. These increases may reflect the
time-dependent development of focally packed areas and the
induction of apoptosis in these areas. Independent studies
demonstrated that caspase activities were basically indistinguishable
in non-treated and ethanol-treated cultures, and directly related to
the confluence of the cultures.2 In contrast, treatment of
1c1c7 (Fig. 4A) or TAHR cells (Fig. 4B) with a
concentration of C2-ceramide (30 µM) that
induced DNA fragmentation increased caspase specific activities
~400-500-fold. The kinetics of caspase activation and DNA
fragmentation paralleled one another in C2-ceramide-treated
1c1c7 and TAHR cultures (compare Fig. 1E with Fig.
4A and Fig. 3L with Fig. 4B).
Caspase specific activities in the AHR-deficient Tao and TCMV cells
were also elevated following exposure to C2-ceramide. However, the level of caspase activation in the two AHR-deficient lines
was less than that measured in the 1c1c7 and TAHR lines (Fig. 4,
A and B). Indeed, caspase specific activities in
C2-ceramide-treated TCMV cells were, at most, only 3-fold
higher than the values measured in solvent-treated cultures. Hence, the
absence of DNA fragmentation in C2-ceramide-treated
AHR-deficient lines correlated with the diminished activation of
caspases (compare Fig. 1F with Fig. 4A and Fig.
3K with Fig. 4B).
Analyses of caspase activation in TAHR and TCMV cells at varied
C2-ceramide concentrations are presented in Fig.
5. No effects were seen in either cell
line at concentrations of Ceramide Induction of Apoptosis in a 1c1c7 Variant Cell Line
Lacking ARNT--
Ligand-activated functions of the AHR are thought to
require its heterodimerization with ARNT (1-3). Although ceramide has not been identified as a ligand of the AHR, we investigated whether the
effects of ceramide require a functional ARNT protein.
BPrc1 cells were derived from the 1c1c7 line and contain
comparable amounts of the AHR, but no functional ARNT (35). Exposure of BPrc1 cells to 60 µM C2-ceramide
(30 µM concentration was not tested) suppressed cell
proliferation (Fig. 6A), and
enhanced permeability to trypan blue (Fig. 6B). Like the
parental 1c1c7 line, BPrc1 cells were more sensitive than
Tao cells to the killing actions of C2-ceramide (Fig.
6B). Exposure of BPrc1 cultures to
C2-ceramide resulted in caspase activation (Fig. 6,
C and D), and the development of nucleosomal DNA
ladders (Fig. 6E), and loss of high molecular weight DNA
(Fig. 6F). Indeed, 1c1c7 and BPrc1 cells were
quite similar to one another with respect to the concentrations of
C2-ceramide required for caspase activation (compare Figs.
5 and 6C) and the magnitude of caspase activation following
C2-ceramide exposure (compare Figs. 4A and
6D).
Effects of Ceramide Analogs and AHR Ligands on Ceramide induced
Apoptosis--
Dihydro-N-acetyl-D-erythro-sphingosine
(DHC2-ceramide) differs from C2-ceramide in
only one respect; it lacks the double bond between carbons 4 and 5 of
the sphingoid backbone. This single alteration drastically alters its
biological activities. Specifically, it does not induce apoptosis in
cells that normally respond to C2-ceramide (39).
Concentrations of DHC2-ceramide
C2-ceramide has dimensions and structural features which
approximate some of the modeled characteristic of AHR ligands (30-33). TCDD is a well characterized agonist of the AHR (1). Exposure of 1c1c7
cultures to a concentration of TCDD that transcriptionally activates
Cyp1a1 (38) had no effects on cell proliferation or viability (Fig. 8, A and
B). Furthermore, preincubation of 1c1c7 cultures with TCDD
did not affect the kinetics of cell killing caused by subsequent
exposure to C2-ceramide (Fig. 8B).
Doxorubicin- and Staurosporine-induced Apoptosis in Variant and
Parental 1c1c7 Cells--
Numerous chemicals are capable of inducing
apoptosis. Staurosporine and doxorubicin, like ceramide, induce caspase
activation through a Bcl-2 inhibitable process that probably involves
the Apaf-1 complex (34). Exposure of 1c1c7 and AHR-deficient Tao cultures to concentrations of doxorubicin
Exposure of 1c1c7 and Tao cultures to concentrations of staurosporine
The murine hepatoma 1c1c7 cell line and its AHR/ARNT variants have
been used extensively as tools in the study of AHR function. Use of
these lines led to the characterization of the AHR as a ligand-activated transcription factor that functions in concert with
ARNT to activate a series of genes having dioxin-responsive elements in
their flanking sequences (1, 35, 36, 45). A recent investigation
employing 1c1c7 cells and AHR variants demonstrated that AHR content,
in the absence of exogenous AHR ligands, affects cell shape, the
duration of the G1 phase of the cell cycle, and the
expression of a hepatic-specific, differentiation-related gene (12).
The current investigation defines a new function for the AHR in cells
of the 1c1c7 lineage. Specifically, within the series of cell lines
surveyed, those having the lowest levels of AHR expression were the
least susceptible to ceramide-induced apoptosis. Thus, susceptibility
to ceramide-induced apoptosis was directly related to the AHR contents
of these cell lines.
One plausible explanation for the differential sensitivities of
AHR-containing and AHR-deficient cell lines to ceramide-induced apoptosis is that ceramide is an AHR ligand. We reasoned that if
C2-ceramide were acting as an AHR ligand, its effects might be duplicated by other known AHR ligands. However, this was not the
case. Neither TCDD nor Although the AHR content of cells of the 1c1c7 lineage influenced
caspase activation by ceramide, it had no affect on the kinetics or
magnitude of caspase activation following exposure to doxorubicin or
staurosporine. Thus, the differential sensitivities of wild type and
AHR-deficient cell lines to ceramide-induced apoptosis reflect a
specific action of ceramide, as opposed to a generalized resistance of
the AHR-deficient lines to apoptotic inducers. These data raise the
issue of whether ceramide signaling, in general, is regulated by AHR
content. Ceramide induces cell cycle arrest in many cell types (for
review, see Ref. 24). This activity reflects a G1 block and
is characterized by the accumulation of hypophosphorylated
retinoblastoma protein (24, 46). We were unable to define a cytostatic,
non-apoptotic concentration of C2-ceramide in 1c1c7 cells.
In contrast, concentrations of C2-ceramide were identified
that arrested both 1c1c7 and TCMV proliferation, which did not induce
apoptosis of the AHR-deficient TCMV cell line (as assessed by DNA
laddering and caspase activation). Hence, AHR content appears to
differentially influence the cytostatic and apoptotic activities of
ceramide. It should be noted that an uncoupling of the cytostatic and
apoptotic activities of C2-ceramide has also been observed
in Raji cells (47), and can be operationally induced in
ceramide-treated Molt-4 and U937 cells by cotreatment with a
cell-permeable diacylglycerol analog or phorbol 12-myristate 13-acetate
(29).
Intracellular ceramide levels are often elevated following
receptor-triggered (e.g. CD95/Apo1 or tumor necrosis
factor- Ceramide affects a variety of signaling molecules and pathways (24). It
is a direct activator of a PPA2-like phosphatase termed
ceramide-activated protein phosphatase (CAPP; Refs. 24 and 53) and a
membrane-bound, Ser/Thr protein kinase designated ceramide-activated
protein kinase (CAP kinase; Refs. 54 and 55). This latter enzyme was
recently shown to be identical to kinase suppressor of Ras (56), and is
capable of activating raf-1 kinase. Ceramide is also an activator of
the Treatment of cells and isolated mitochondria with ceramide increases
mitochondrial production of reactive oxygen species (ROS; 28, 61). The
ROS arise as a consequence of ceramide inhibition of complex III,
blockage of respiration, and the subsequent diversion of electrons from
ubisemiquinone to molecular oxygen (61, 62). The ROS, in turn, lead to
the development of oxidative stress, mitochondrial damage, and
disruption of mitochondrial membrane potential and permeability (61,
63). Such a scenario could lead to the release of cytochrome
c, which is required for caspase activation by the
mitochondrial-associated Apaf-1 complex (64). In preliminary studies we
have found that C2-ceramide exposure of suspended 1c1c7
cells results in a concentration-dependent suppression of
respiration.3 In contrast,
the effects of ceramide on respiration appear to be muted in Tao
cells.3 These observations may provide a basis for the
observed lack of caspase activation in the AHR-deficient cells. We are
currently investigating this line of research.
Nucleosomal DNA fragmentation is a late stage marker of apoptotic
cells, and is catalyzed by a 40-kDa DNase designated DNA fragmentation
factor (DFF40; Refs. 65-67). DFF40 normally exists in the cytosol in
an inactive form as a consequence of its association with DFF45 (66,
66). In vitro studies in which DFF40 and DFF45 are mixed
with caspase-3 (66, 67) and investigations employing caspase-3-deficient cells (68) and caspase inhibitors (69-71) collectively demonstrate that caspase-3 activates DDF40 by cleaving DFF45, which facilitates dissociation of the heterodimeric complex. Given the role of caspase-3 in activating DFF40, and the inability of
C2-ceramide to activate caspase-3 in our AHR-deficient cell lines, it is easy to rationalize the absence of DNA laddering in these
cells following C2-ceramide exposure.
Although C2-ceramide-treated AHR-deficient 1c1c7 variant
cell lines did not exhibit characteristics commonly associated with apoptotic cells (morphology, caspase-3 activation, DNA laddering), they
were killed by ceramide, as determined by measurements of trypan blue
exclusion. The sensitivities of the parental and AHR-deficient variant
cell lines to the cytotoxic effects of C2-ceramide,
however, were markedly different. At comparable concentrations of
C2-ceramide, a lower percentage of AHR-deficient cells
died, and the kinetics of cell killing were considerably slower than
those observed in their AHR-containing counterparts. Hence, the
mechanisms by which C2-ceramide killed 1c1c7 cells and its
AHR-deficient variants are probably different. Additional studies are
required to determine if the AHR-deficient variants die by a necrotic
pathway following C2-exposure. It should be emphasized that
other examples exist documenting cell killing by apoptotic agents via a
pathway that does not lead to the development of features
characteristic of apoptotic cells. Specifically, MCF-7 cells do not
express functional caspase-3 as a consequence of a 47-base pair
deletion within exon 3 of the CASP-3 gene (68).
Nevertheless, numerous apoptotic agents kill MCF-7 cells by a process
that neither involves caspase-3 or DFF40 activation nor results in the
morphological characteristics of apoptotic cells (see Ref. 68, and
references therein). In this respect, the effects of apoptotic agents
on MCF-7 cells are very similar to the effects of
C2-ceramide on AHR-deficient 1c1c7 variant lines.
In summary, our studies define a new role for the AHR, which is related
to its regulation of ceramide-initiated apoptosis. The extent to which
the AHR regulates the numerous signaling pathways affected by ceramide
is not known. However, there appears to be some specificity since
ceramide was cytostatic, but not apoptotic to AHR-deficient cells of
the 1c1c7 lineage. Furthermore, our studies are the first to define a
biological function for the AHR that is independent of its interaction
with ARNT, and its functioning as a ligand-activated, ARNT-associated
transcription factor.
-naphthoflavone) neither induced apoptosis nor modulated the
development of apoptosis in C2-ceramide-treated 1c1c7
cultures. AHR content did not affect staurosporine- or
doxorubicin-induced apoptosis. These results suggest the AHR modulates
aspects of ceramide signaling associated with the induction of
apoptosis but not cell cycle arrest, and does so by a mechanism that is independent of its interaction with aryl hydrocarbon receptor nuclear
translocator and exogenous AHR ligands.
INTRODUCTION
Top
Abstract
Introduction
References
) that causes hepatocyte
apoptosis (22). Presumably, absence of the AHR facilitates conditions
that lead to the activation of latent transforming growth factor-
(22).
EXPERIMENTAL PROCEDURES
-Naphthoflavone, doxorubicin, and
staurosporine were purchased from Sigma. TCDD was the gift of Dr. S. Safe (Texas A & M University, College Station, TX).
N-Acetyl-D-erythro-sphingosine (C2-ceramide) and
dihydro-N-acetyl-D-erythro-sphingosine
(DHC2-ceramide) were purchased from Calbiochem-Novabiochem
Corp. (San Diego, CA). Ac-DEVD-AMC and 7-amino-4-methyl-coumarin (AMC)
were obtained from PharMingen (San Diego, CA) and Aldrich,
respectively. Geneticin,
-minimal essential medium, fetal bovine
serum, and penicillin-streptomycin were purchased from Life
Technologies, Inc.
-minimal
essential medium containing 5% fetal bovine serum and 100 units/ml
penicillin and 100 µg/ml streptomycin. The TCMV, TAHR, WCMV, and WARV
lines were maintained in medium containing 500 ng/ml Geneticin.
-naphthoflavone, doxorubicin, or staurosporine (dissolved in
Me2SO). At various times after treatment, culture medium
was lightly sprayed over the culture surface to dislodge loosely
attached cells, which were transferred to a centrifuge tube. Attached
cells were removed with a 0.25% trypsin-EDTA mixture and added to the
centrifuge tube. In some instances the pooled cell suspensions were
pelleted by centrifugation and resuspended in phosphate-buffered
saline. Cells were counted with a hemacytometer. Viability was assessed
by trypan blue exclusion. Each sample was counted a minimum of three times.
70 °C. On the day of assay, lysates were thawed on ice,
homogenized for 20 s (PT 1200 Polytron, Kinematica, Switzerland),
and centrifuged at 18,000 × g for 12 min at 4 °C. Supernatant fluids were kept on ice until assayed by a modified version
of a protocol supplied by the provider of Ac-DEVD-AMC (PharMingen).
Assays were performed at room temperature and initiated by the addition
of 100 µl of supernatant fluid to 1.9 ml of protease assay buffer
(final concentrations: 20 mM Hepes, pH 7.4, 10% glycerol, 2 mM dithiothreitol, and 20 µM Ac-DEVD-AMC).
AMC release was monitored with a Perkin-Elmer LS-5 fluorescence
spectrophotometer using an excitation wavelength of 380 nm and an
emission wavelength of 460 nm. A standard curve created with AMC was
used to convert fluorescence intensity into picomoles of product
produced in the presence of supernatant fluids. Protein concentrations
were determined with Bio-Rad protein assay reagent using bovine serum
albumin as a standard. Caspase activities are reported as nanomoles of AMC released/min/mg of protein of supernatant fluid. Initial
characterization studies demonstrated that substrate cleavage was
linear with time and directly proportional to the amount of protein in
the assay.2
70 °C. Non-adherent cells in the culture medium were recovered by
centrifugation and also stored at
70 °C.
-mercaptoethanol, and 0.5% sarkosyl.
Lysates were transferred to the tubes containing pellets of
non-adherent cells. Lysates were extracted with 5 ml of
Tris-equilibrated phenol (pH >7.6) plus 1 ml of chloroform:isoamyl
alcohol (49:1). After centrifugation for 1 h at 6500 × g at 4 °C, the aqueous phase was transferred to new
tubes. Nucleic acids were precipitated by addition of an equal volume
of isopropanol and subsequently pelleted by centrifugation. Pellets
were resuspended in 0.5 ml of 10 mM Tris, 1 mM
EDTA, pH 8.0 (TE), 0.1% SDS, and 40 µg/ml RNase A and incubated at
37 °C, for 1 h. After successive extractions with
Tris-equilibrated phenol (pH >7.6):chloroform:isoamyl alcohol
(25:24:1) and chloroform:isoamyl alcohol (24:1), DNA was precipitated
by the addition of 1/50 volume of 5 M NaCl and 2 volumes of
95% EtOH. DNA was pelleted by centrifugation, resuspended in TE (~10
µl per 1 × 106 cells), and quantified by
spectrophotometry. DNAs were mixed with ethidium bromide (80 ng/ml) and
separated on 2% agarose, 1× TAE gels. DNA was visualized under UV
light and recorded with a IS-1000 Digital Imaging System (Alpha
Innotech Corp., San Leandro, CA). Nucleosomal ladders were visualized
with exposures of ~2 s. Briefer exposures (
1/15 s) were used to
visualize high molecular weight DNA.
RESULTS
30 µM completely suppressed cell division (Fig.
1A), enhanced cell permeability to trypan blue (Fig.
1C), and caused dramatic morphological changes.
Specifically, within 4-6 h of exposure to
30 µM
C2-ceramide, the cells began to vacuolate, and by 12-16 h
had shrunken, as had the nuclei.2 DNA fragmentation and the
development of nucleosomal ladders, a characteristic of late stage
apoptotic cells, were detected within 16 h of
C2-ceramide treatment (Fig. 1E) and preceded
alterations in membrane permeability to trypan blue (Fig. 1, compare
C and E).
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Fig. 1.
Ceramide-induced apoptosis in 1c1c7 and Tao
cells. Suspensions of 1c1c7 (first column)
and Tao (second column) were plated 1-2 days
prior to onset of treatment. At time zero cultures were treated with
Me2SO ( ) or varying concentrations of
C2-ceramide (
, 1 µM;
, 10 µM;
, 30 µM;
, 60 µM;).
Cultures were harvested at various times after treatment for analyses
of cell numbers (panels A and B,),
cell survival (panels C and D), and
isolation of DNA. To facilitate comparison, the 60 µM
concentration from panel D is indicated in
panel C by a dashed line. Purified DNA (24 µg)
from the cultures treated with 30 µM
C2-ceramide for indicated times was mixed with ethidium
bromide and separated on 2% agarose gels. High molecular weight DNA
was detected with UV and photographed using a 1/30th-s exposure
(panels G and H). Fragmented DNA on
the same gel was photographed using a 2-s exposure (panels
E and F). C-48 denotes
Me2SO-treated cultures harvested at the same time as the
48-h ceramide treatment group (panels E-H). Data
in panels A-D represent means ± S.E. of
3-6 plates.
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Fig. 2.
AHR content and TCDD responsiveness of 1c1c7
cells and variant lines. Exponentially growing cultures were
harvested and processed for AHR protein analyses without any treatment
(top panel) or were exposed to Me2SO
or 2 nM TCDD for 6 h prior to being harvested for
isolation of RNA and analyses of CYP1A1 and 7 S RNAs (bottom
panel). Analyses were made on 30 µg of protein
(top panel) or 10 µg of total RNA
(bottom panel).
30 µM (Fig.
1B). However, the killing effects of anti-proliferative
concentrations of C2-ceramide were muted in Tao cells (Fig.
1D). Specifically, the kinetics of killing were slower and
the absolute level of killing was much less than that seen in parental
1c1c7 cells (Fig. 1, compare C and D). Indeed, ~30% of C2-ceramide-treated Tao cells were viable after
even 4 days of treatment (n = 3 experiments).2 Furthermore, at comparable loadings of DNA,
nucleosomal ladders were not detected in Tao cells following 48 h
(Fig. 1, compare E and F), or even 72 h of
exposure to C2-ceramide.2
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Fig. 3.
Ceramide-induced apoptosis in transfected
1c1c7 and Tao cells. Suspensions of WCMV (first
column), WARV (second column), TCMV
(third column), and TAHR (fourth
column) were plated 1-2 days prior to onset of treatment.
At time zero, cultures were treated with ethanol ( ) or varying
concentrations of C2-ceramide (
, 1 µM;
, 30 µM). Cultures were harvested at various times
after treatment for analyses of cell numbers (panels
A-D), cell survival (panels E-H),
and isolation of DNA. To facilitate comparison, the 30 µM
concentrations from panels F and G are
indicated by dashed lines in panels
E and H, respectively. Analyses of fragmented DNA
(panels I-L) and high molecular weight DNA
(panels M-P) are as described in the legend of
Fig. 1. Data in panels A-H represent the
means ± S.E. of 3-6 plates.
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Fig. 4.
Kinetics of caspase activation in 1c1c7 and
1c1c7 variant cell lines. Suspensions of 1c1c7, Tao, TAHR, and
TCMV cells were plated 2 days prior to treatment with ethanol or 30 µM C2-ceramide. Cultures were harvested at
various times after treatment for analyses of caspase activities. Data
represent the means ± S.E. of analyses performed on 3 plates.
A: , 1c1c7-C2;
, Tao-C2;
,
1c1c7-EtOH;
, Tao-EtOH. B:
, TAHR-C2;
,
TCMV-C2;
, TAHR-EtOH;
, TCMV-EtOH.
10 µM. In TAHR cells a small
activation was observed at 20 µM, and near-maximal activation occurred at 30 µM. In marked contrast, no
activation occurred in the AHR-deficient TCMV line at concentrations up
to 60 µM C2-ceramide (Fig. 5). Similar
results were obtained in analyses of the 1c1c7 and Tao
lines.2 Specifically, caspases were not activated in Tao
cells treated with concentrations of C2-ceramide as high as
60 µM, whereas maximal activation occurred at 30 µM in 1c1c7 cells. Thus, the differential responsiveness
of AHR-containing and AHR-deficient cell lines to
C2-ceramide did not reflect a shift in the
concentration-response curves.
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Fig. 5.
Caspase activation in TCMV and TAHR cells as
a function of C2-ceramide concentration. Suspensions
of TAHR ( ) and TCMV cells (
) were plated 2 days prior to
treatment with varied concentrations of C2-ceramide.
Cultures were harvested 26 h after treatment for analyses of
caspase activities. Data represent the means ± S.E. of analyses
performed on 3 culture dishes.
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Fig. 6.
Ceramide-induced apoptosis in
BPrc1 cells. Suspensions of BPrc1 were
plated 1-2 days prior to onset of treatment. At time zero, cultures
were treated with solvent or varying concentrations of
C2-ceramide (see symbols in figure). Cultures were
harvested at various times after treatment for analyses of cell numbers
(panel A), survival (panel
B), caspase activation (panels C and
D), and DNA. Analyses of fragmented DNA (panel
E) and high molecular weight DNA (panel
F) are as described in the legend of Fig. 1. C-48
denotes Me2SO-treated cultures harvested at the same time
as the 48-h ceramide treatment group. Data in panels
A-D represent means ± S.E. of 3-6 plates. To
facilitate comparison to an AHR-deficient cell line, the 60 µM C2-ceramide survival data for the Tao cell
line from Fig. 1D is indicated in panel
B by a dashed line.
60 µM
neither affected 1c1c7 proliferation nor viability (Fig.
7, A and B,
respectively). These data suggest that the apoptosis seen in 1c1c7
cells following C2 exposure represents a ceramide-specific
process, as opposed to a nonspecific lipid effect.
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Fig. 7.
Dihydro-N-acetyl-D-erythro-sphingosine-induced
apoptosis. Suspensions of 1c1c7 cells were plated 1 day prior
to treatment with either solvent or DHC2-ceramide. Cultures
were harvested for analyses of cell numbers (panel
A) and survival (panel B) at various
times after treatment. Data represent the means ± S.E. of
analyses performed on 3 culture dishes. , ethanol;
, 30 µM DHC2;
, 60 µM
DHC2.
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Fig. 8.
Effects of AHR ligands on ceramide-induced
apoptosis. Suspensions of 1c1c7 cells were plated 1 day prior to
treatment with either solvent or C2-ceramide and/or TCDD
(left column) or C2-ceramide and/or
-NF (right column). Cultures were treated with
AHR ligands 2 h prior to the addition of C2-ceramide.
Solvent controls and cultures treated with only C2-ceramide
were also pretreated with solvent in order to mimic the double solvent
exposure occurring in cultures treated with C2-ceramide + AHR ligand. Cultures were harvested at various times after ceramide
treatment for analyses of cell numbers (panels A
and C) and survival (panels B and
D). Data represent the means ± S.E. of analyses
performed on 3 culture dishes. A and B:
,
Me2SO;
, 30 µM C2;
, 60 µM C2;
, 5 nM TCDD;
, 30 µM C2 + 5 nM TCDD;
, 60 µM C2 + 5 nM TCDD. C
and D:
, Me2SO;
, 1 µM
-NF;
, 30 µM C2;
, 1 µM
-NF + 30 µM C2.
-Naphthoflavone (
-NF) is also a ligand of the AHR. However, it
functions as an antagonist in many cell types at low micromolar concentrations, and as an agonist at concentrations of
10
µM (42-44). An antagonist concentration of
-NF was
weakly cytostatic to 1c1c7 cultures (Fig. 8C), but had no
effect on cell viability (Fig. 8D). Incubation of 1c1c7
cultures with
-NF prior to treatment with C2-ceramide
affected neither the kinetics nor magnitude of cell killing caused by
C2-ceramide (Fig. 8D).
0.1 µM
strongly suppressed the proliferation of both cell lines (Fig.
9, A and B). Based upon analyses of trypan blue permeability at various concentrations of
doxorubicin, Tao and 1c1c7 cultures appeared to be fairly similar in
their sensitivities to the killing actions of the drug (Fig. 9,
E and F). Indeed, the kinetics of caspase
activation and levels of activation, at each concentration of
doxorubicin tested, were very similar in the two cell lines (Fig. 9,
compare I and J).
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Fig. 9.
Staurosporine- and doxorubicin-induced
apoptosis in 1c1c7 and Tao cells. Suspensions of 1c1c7 and Tao
cells were plated 1 or 2 days prior to treatment with
Me2SO, or varied concentrations of doxorubicin or
staurosporine. Cultures were harvested at various times after treatment
for analyses of cell numbers (panels A-D),
survival (panels E-H), and caspase activities
(panels I-L). Data represent the means ± S.E. of analyses performed on three culture dishes. A,
B, E, F, I, and
J: , Me2SO;
, 0.01 µM;
,
0.1 µM;
, 1 µM;
, 10 µM. C, D, G,
H, K, and L:
,
Me2SO;
, 5 nM;
, 20 nM;
, 100 NM.
5 nM totally suppressed the proliferation of both cell lines (Fig. 9, C and D). Although analyses of
trypan blue exclusion suggested that Tao cultures were slightly less
susceptible than 1c1c7 cultures to the killing actions of low
concentrations of staurosporine, this differential response was lost at
a concentration of 100 nM staurosporine (Fig. 9, compare
G and H). Furthermore, analyses of caspase
activation suggested no differences in the responsiveness of 1c1c7 and
Tao cultures to staurosporine. The kinetics and magnitude of caspase
activation, at each concentration of staurosporine tested, were very
similar in the two cell lines (Fig. 9, compare K and
L).
DISCUSSION
-NF triggered the induction of apoptosis in
1c1c7 cells or modulated the induction of apoptosis by ceramide (see
Fig. 8). Reciprocally, we found that treatment of 1c1c7 cultures with
concentrations of C2- or C6-ceramide of
60
µM neither reproducibly elevated CYP1A1 mRNA contents
(a 2.5-fold elevation was noted at 4 h after treatment in only one
of five experiments), nor affected the transcriptional activation of
Cyp1a1 by TCDD.2 These latter studies
demonstrate that ceramide is not an AHR antagonist. Although our
studies do not rule out the possibility that ceramide affects the AHR
by some indirect mechanism, they suggest that ceramide is not an AHR
ligand. In addition, ceramide-induced apoptosis occurred in a variant
of 1c1c7 cells (e.g. BPrc1 cell line) having
parental AHR contents, but no functional ARNT. This is an exciting
finding, as it demonstrates that the mechanism by which the AHR
modulates ceramide-induced apoptosis can not involve ligand-activated
AHR/ARNT heterodimer. To the best of our knowledge, this is the first
demonstration of an AHR-modulated process that does not require ARNT.
) and non-receptor-triggered (e.g. ionizing
radiation, staurosporine, daunorubicin, etc.) apoptosis (24, 25, 48,
49). These elevations can reflect either de novo
biosynthesis (49) or sphingomyelinase activation and degradation of
sphingomyelin (25). Because exogenously added, cell-permeable ceramides
induce apoptosis in several cell types, it has been hypothesized that
ceramide produced in situ may be the initiator of the
execution phase of apoptosis. This hypothesis is supported by two
additional lines of experimentation. First, lymphoblasts and
fibroblasts derived from humans (e.g. individuals suffering
from Neimann-Pick disease) or mice defective in acidic sphingomyelinase
are resistant to ionizing radiation or doxorubicin induced-apoptosis,
but apoptose if treated with an exogenous source of ceramide (48, 50).
Second, some multiple drug-resistant cell lines express a
glucosylceramide synthase that inactivates ceramide via conjugation
(51, 52). Treatment of such lines with an inhibitor of glucosylceramide
synthase renders them sensitive to the apoptotic actions of
cell-permeable ceramide analogs and a variety of chemotherapeutic
agents (52). Nevertheless, the observed sensitivity of the
AHR-deficient Tao cells to exogenously added doxorubicin or
staurosporine, but not C2-ceramide, strongly suggests that
the first two agents activate caspases by a ceramide-independent process. The 1c1c7 and AHR variant lines may be useful tools for the
characterization of this process, and assessment of the contributions of ceramide to the initiation of apoptosis by apoptotic inducers. Meaningful interpretation of comparisons of the sensitivities of wild
type and AHR "knockout" mice to the actions of apoptotic agents, or
physiological processes that entail an apoptotic process (e.g. regulation of T cell proliferation following
stimulation) will be dependent upon such information.
isozyme of protein kinase C (57, 58) and members of the
stress-activated protein kinase (SAPK) cascade (27, 59). It is also a
potent inhibitor of complex III of the mitochondrial respiratory chain (60). Of these various effects, ceramide activation of the SAPK cascade
and inhibition of complex III activity have been linked to the
induction of apoptosis (27, 28, 59, 61).
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ACKNOWLEDGEMENT |
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We thank Dr. Cornelis Elferink for introducing the idea that ceramide may be an AHR ligand.
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FOOTNOTES |
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* This work was supported in part by National Institutes of Health Grant CA34469, by Pilot Project Grant MCT26 awarded by P30 ES06639, and by assistance from the services of the Cell Culture Facility Core and Cell Imaging and Cytometry Facility Core, which are supported by National Institutes of Health NIEHS Grant P30 ES06639.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.
To whom correspondence should be addressed: Inst. of Chemical
Toxicology, Wayne State University, 2727 Second Ave., Rm. 4000, Detroit, MI 48201. Fax: 313-577-0082; E-mail:
john.reiners.jr{at}wayne.edu.
The abbreviations used are:
AHR, aryl
hydrocarbon receptor; -NF,
-naphthoflavone; Ac-DEVD-AMC, acetyl-Asp-Glu-Val-Asp-7-amino-4-methylcoumarin; AMC, 7-amino-4-methyl-coumarin; ARNT, aryl hydrocarbon receptor nuclear
translocator; C2-ceramide, N-acetyl-D-erythro-sphingosine; DFF, DNA fragmentation factor; DHC2-ceramide, dihydro-N-acetyl-D-erythro-sphingosine; ROS, reactive oxygen species; TCDD, 2,3,7,8-tetrachlorodibenzo-p-dioxin.
2 R. E. Clift and J. J. Reiners, Jr., unpublished observations.
3 B. Taffe and J. J. Reiners, Jr., unpublished observations.
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REFERENCES |
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