(Received for publication, October 9, 1996, and in revised form, April 24, 1997)
From the Retinoic acid (RA) and 9-cis-RA
induce growth arrest and differentiation of S91 melanoma cells. RA
activates retinoic acid receptors (RARs), whereas 9-cis-RA
activates both RARs and retinoid X receptors (RXRs). Both classes of
receptors function as ligand-dependent transcription
factors. S91 melanoma cells contain mRNA for RXR Retinoid receptors, which include retinoic acid
(RA)1 receptors (RARs) and retinoid X
receptors (RXRs), are members of a large group of
ligand-dependent transcription factors (1, 2). There are
three separate genes ( S91 murine melanoma cells constitutively express RAR We set out to answer these questions by using a number of RXR and RAR
isoform-specific agonists to evaluate their effects on cell growth,
RAR Cell Culture and Cell Proliferation Assay
All tissue culture plates and flasks were from Costar
(Cambridge, MA). S91 cells (ATTC CCL 53.1) were grown in Dulbecco's modified Eagle's medium with 10% (v/v) fetal calf serum at 37 °C in 5% CO2 in humidified air. Retinoid stock solutions (10 or 1 mM in Me2SO) were added to media to
achieve the desired concentration. Final Me2SO
concentrations in cell culture medium never exceeded 0.1% (v/v). For
the cell proliferation assay, cells were seeded at a density of about
7,500 cells/well in triplicate in 96-well plates, and treated for 5 days in total with the indicated retinoids. Media with/without
retinoids was changed every 24 h. At the fifth day, relative
viable cell numbers were obtained using the CellTiter 96TMAQueous Non-Radioactive Cell Proliferation
Assay kit from Promega (Madison, WI). Absorbance at 490 nm was
determined in a UV max kinetic microplate reader (Molecular Devices
(Sunnyvale, CA). Morphology of cells was monitored on an Olympus IMT-2
phase-contrast microscope, and photographed through an attached Olympus
OM-2s program camera (Tokyo, Japan) on Kodak T-Max 400 film (Rochester, NY).
Plasmids, Transfection, and Luciferase/ A plasmid with a 4.4-kilobase chromosomal fragment containing
the RAR Northern Blot Analysis
Cells were grown in 150-cm2 plates until 75%
confluent. Medium was changed containing the appropriate amounts of
retinoid or control (Me2SO), and/or 10 µg/ml
cycloheximide (Sigma). Cells were harvested after 8 h, washed
twice with phosphate-buffered saline, and poly(A)+ mRNA
extracted using a Qiagen Direct mRNA Midi Kit (Chatsworth, CA). Two
µg of mRNA was subjected to electrophoresis through a denaturing
formaldehyde-agarose gel (1%). Blotting procedures, hybridization/washing conditions, preparation of radioactively labeled
probe, and quantitation were all performed as described (25).
Cyclophilin probe serves as internal control for loading and blotting,
and used to normalize values obtained from the RAR Melanin Assay
Cells were grown in 10-cm plates, and treated for 5 days with
the required amount of indicated retinoids or control
(Me2SO). Medium with/without retinoids was changed every
24 h. Adherent cells were harvested by trypsinization and washed
twice with phosphate-buffered saline. 2 × 104 cells
were pelleted and lysed overnight in 100 µl, 1 N KOH at 80 °C, and absorbance at 490 nM was determined.
Apoptosis Assays
Cells were grown until 50% confluent
in 10-cm plates and treated for 24 h with the indicated retinoids.
4 × 106 cells (adherent and floating) were harvested
by trypsinization, washed twice in phosphate-buffered saline, and
pellets were lysed in 400 µl of lysis buffer (10 mM EDTA,
50 mM Tris, pH 8.0, 0.5% (w/v) Sarcosyl, 0.5 mg/ml
proteinase K) for 3 h at 50 °C. Next, protein was removed by
treatment with an equal volume of phenol/chloroform/isoamyl alcohol
(50:48:2), and once with chloroform/isoamyl alcohol (24:1). DNA was
precipitated, dissolved in 100 µl of TE buffer with 0.25 mg/ml RNase
A, and incubated at 50 °C for 1 h followed by 1 h at
37 °C. 25 µl were then loaded onto a 1.5% agarose gel in TAE buffer with 0.5 µg/ml ethidium bromide and separated by
electrophoresis. DNA was visualized by UV light at 305 nM
and photographed.
Cells were grown in 6-well plates, and adherent and floating
cells were harvested by trypsinization, washed twice in
phosphate-buffered saline, and counted. Pelleted cells were then
treated following the manufacturer's protocol with the Cell Death
Detection ELISA (Boehringer Mannheim, Indianapolis, IN). 1,000 cells
were used in the enzyme-linked immunosorbent assay, and
absorbance was determined at 405 nM in a UV max kinetic
microplate reader (Molecular Devices, Sunnyvale, CA).
Binding Assay
Equilibrium dissociation constants (Kd
values) for the interaction of the different retinoids with the three
RAR isoforms were determined in vitro at 4 °C by
measuring displacement of radiolabeled reference retinoid CD367 by
increasing dose of non-radioactive ligands. The assays were performed
as described before, using RARs that were obtained by overexpression in
COS-7 cells (35). At least three independent determinations for binding were performed. The average standard error of the mean was within 25%.
Kd values and RXR-specific transactivation
properties of the pan-RXR ligand CD2624 were described previously (36). Except for CD2624, none of the compounds listed in Table I have significant binding affinity for RXR, nor do they transcriptionally activate this receptor (not shown).
Table I.
Overview and binding specificities of retinoids for RXR and RAR
isoforms used in these studies
Department of Surgery,
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES
, RXR
,
RAR
, RAR
, and RAR
in low levels. Among these, only RAR
gene
transcription is induced by retinoids. However, at present the
individual role(s) for each RXR and RAR isoform in these processes is
unclear. We assessed the function of all isoforms in the S91 melanoma
model by using RXR and RAR isoform-specific retinoids to study their
effects on cell growth, RAR
expression, and differentiation. Activation of each of the endogenous RXR or RAR isoforms induces RAR
gene expression, and blocks cellular proliferation. However, only the
RAR
-ligands cause additional differentiation toward a melanocytic
phenotype, which coincides with substantial apoptosis well before
morphological changes are apparent. Apoptosis is completely dependent
on de novo protein synthesis but cannot be induced by changes in activities of AP-1, protein kinase C, and protein kinase A,
nor can it be blocked by the presence of the antioxidant glutathione. These results argue against a specific role for RAR
, but suggest that RAR
has a critical role in a genetic switch between melanocytes and melanoma, and induction of ligand-dependent
apoptosis.
,
, and
) for each class of retinoid
receptors, respectively, and multiple splice variants exist (3, 4).
RARs are activated by RA and its metabolized stereoisomer,
9-cis-RA (5, 6), whereas RXRs only bind the latter.
Ligand-bound receptors can modulate the expression of genes containing
appropriate response elements (RA response element, RARE). A productive
RARE generally consists of the direct repeat AGGTCA spaced by two or
five nucleotides (DR2 and DR5, respectively), although other functional
configurations exist (Ref. 1, and references therein). RXRs activate
through retinoid X response elements (RXREs), which resemble RAREs
except that the spacing for an optimal response is one nucleotide (DR1)
(7-9). It is generally assumed that RARs are heterodimerized with RXRs
inside the cell (7, 8, 10-16), but RXRs can also form homodimers in
the presence of its own ligand, 9-cis-RA (8, 9). RA
especially is known to have profound effects on vertebrate development
and differentiation in vivo (17, 18), and it can also induce
differentiation of a large number of tumor cell lines in
vitro (19). Thus, RARs and RXRs are at the peak of a pyramid of an
extremely complex genetic network to control cell growth and
differentiation. Conceivably, mutations or aberrant expression levels
of these receptors might lead to neoplasia (20-22).
, RAR
,
RXR
, RXR
, and very low levels of RAR
(23-25). Upon treatment with RA or 9-cis-RA, a reversible conversion of malignant
melanoma into a benign, melanocytic phenotype takes place suggesting
that a specific genetic program is induced and maintained by RARs
and/or RXRs (23, 25-27). Administration of either of these two
retinoids results in rapid up-regulation of RAR
expression, followed
by cessation of cell division, and morphological differentiation (23-25). A correlation was observed between
retinoid-dependent induction of the RAR
gene and growth
arrest, indicating that RAR
might be involved (23). The RAR
promoter contains a typical RARE (
RARE) of the DR5 type (28-30),
but at least in S91 cells, also appears to be regulated by RXR (25).
Unfortunately, it is difficult to assess the importance of individual
RAR isoforms and RXRs in the above described processes using pan-RAR
and pan-RAR-RXR agonists like RA and 9-cis-RA, respectively.
A more detailed comprehension of the underlying molecular events
leading to neoplastic change in this model would be advantageous to our
basic understanding of cancer, and it might benefit the development of
improved therapies (31).
expression, and morphological differentiation. Our results
suggest that all receptors can induce growth arrest and transcriptional
activation of the RAR
gene. In contrast, only the RAR
ligands
cause morphological differentiation which, interestingly, occurs
concomitantly with substantial apoptosis well before phenotypic changes
become apparent. The mechanism of apoptosis is dependent on newly
synthesized proteins, suggesting that new gene transcription is a
necessary prerequisite. Changes in activities of AP-1, PKC, or PKA do
not lead to apoptosis, whereas high concentrations of the free oxygen
radical scavenger glutathione (GSH) do not block RAR
agonist-induced
cell death. These results suggests that redundant mechanism(s) inhibit
cellular proliferation and RAR
expression. Activation of the RAR
promoter and/or receptor is not a necessary condition for growth arrest
in these cells. It also shows that there is no default mechanism
leading to phenotypic differentiation after growth arrest. Instead,
only RAR
appears to be able to activate the differentiation program,
and induce ligand-dependent apoptosis.
-Galactosidase
Assay
2 promoter linked to the luciferase (Luc) gene,
pW1RAR
2pr-lucif (32), was kindly provided by Dr. E. Linney with
permission of Dr. P. Chambon. Dr. Chambon also provided us with
pSGRAR
2 (33). Plasmids were grown in Escherichia coli
strain DH5
in LB media with ampicillin, and purified using a Qiagen
plasmid maxi kit (Chatsworth, CA). For the transfection with
pW1RAR
2pr-lucif, cells were grown in large flasks (165 cm2) until 75% confluent. Per flask, cells were harvested
by trypsinization, washed in ice-cold Dulbecco's modified Eagle's
medium, resuspended in 0.5 ml of ice-cold Dulbecco's modified Eagle's
medium in a 0.4-cm electroporation cuvette (Invitrogen, San Diego, CA),
and incubated for 5 min on ice with 40 µg of pW1RAR
2pr-lucif and 12 µg of CH110 (Pharmacia Biotech Inc., Piscataway, NJ). CH110 constitutively expresses
-galactosidase activity which serves as an
internal control for the transfection efficiency. Cells were then
electroporated using a Electroporator II (Invitrogen, San Diego, CA)
set at 200 V, 1000 microfarads, incubated on ice for 10 min, and
resuspended in Dulbecco's modified Eagle's medium, 10% fetal calf
serum at 37 °C, and plated on a 6-well plate. After 36 h, cells
were treated with retinoids and harvested 12 h later. Luciferase
assays and
-galactosidase assays were performed as described (34).
Values from the luciferase assay were normalized for
-galactosidase
activity. Fold induction means normalized luciferase activity in the
presence of retinoid/normalized luciferase activity in the absence of
retinoid (control, Me2SO).
probe.
Fold-induction means normalized value of RAR
probe (in the presence
of retinoid and presence or absence of cycloheximide)/value of RAR
probe (in the absence of retinoid and absence of
cycloheximide).
Retinoid
Receptor binding
affinity (Kd/nM)
Primarily
selective for
RAR
RAR
RAR
RA
16
7
3
RAR
,
,
Am80
62
280
816
RAR
Am580
8
131
450
RAR
CD417
6500
36
426
RAR
CD2314
>3757
195
NB
RAR
CD437
6500
2480
77
RAR
CD2325
1144
1245
53
RAR
CD2624a
>5652
NBb
>6208
RXR
,
,
a
Kd values for RXRs, sec Ref. 36.
S91 cells do not express RXR (25).
b
NB, no binding.
Electrophoretic Mobility Shift Assay (EMSA)
RAR and RXR
were obtained by translation in
vitro in the TNT coupled reticulocyte lysate system according to
the manufacturers protocol (Promega, Madison, WI). EMSA conditions were
essentially performed as described previously (25, 34).
32P-End-labeled dsDNA oligonucleotide (
RARE:
61/
29,
5
-AGCTTCCGGGAAGGGTTCACCGAAAGTTCACTCGCATA, 3
-AGGCCCTTCCCAAGTGGCTTTCAAGTGAGCGTATTCGA) was purified over a Nick
column (Pharmacia Biotech Inc.). Reaction mixture, containing 3 µl of
each required lysate (complemented where necessary with unprogrammed
lysate to maintain equal volumes of lysate) and 1 µg of salmon sperm
DNA, with or without 2 µl of recombinant purified AP-1/c-Jun
(Promega) and 250 fmol of double stranded unlabeled competitor
oligonucleotide
RARE or
12-O-tetradecanoylphorbol-13-acetate response element
(AP-1-binding site, 5
-CGCTTGATGAGTCAGCCGGAA, 3
-GCGAACTACTCAGTCGGCCTT,
Promega, Madison, WI) and ligand (RA or CD437 at 1 µM),
was incubated for 15 min at room temperature. Next, radioactive probe
was added (100,000 cpm, 5 fmol) and incubated for another 30 min, and
kept on ice for 10 min before electrophoresis on 4% polyacrylamide
gel.
Retinoids
The chemical nomenclature for the synthetic ligands is: Am80,
4-((5,6,7,8-tetrahydro-5,5,8,8-tetramethyl-2-naphthalenyl)carbamoyl) benzoic
acid (37); Am580,
4-((5,6,7,8-tetrahydro-5,5,8,8-tetramethyl-2-naphthalenyl)carboxamido)benzoic acid (37); CD417,
6-(3-tert-butyl-4-methoxyphenyl)-2-naphthoic acid (38);
CD2314,
2-(5,6,7,8-tetrahydro-5,5,8,8-tetramethyl-2-anthracenyl)-4-thiophene carboxylic acid (90); CD437 (SR11248, AHPN),
6-(3-(1-adamantyl)-4-hydroxyphenyl)-2-naphthoic acid; CD2325,
4-((E)-2-(3-(1-adamantyl)-4-hydroxyphenyl)-1-propenyl)benzoic acid
(38); CD2624,
4-(3,5,5,8,8-pentamethyl-5,6,7,8-tetrahydro-2-naphthylthio)benzoic acid
(91). RA was obtained from Sigma. All other retinoids were synthesized
at CIRD Galderma (Valbonne, France). All retinoids were kept as 30 mM stock solutions in Me2SO, and kept at
20 °C to
80 °C.
For the studies described
here, we selected the following synthetic retinoids (listed in Table
I), and determined their Kd values
for each RAR isoform (35): Am80 and Am580, which have a strong
preference for RAR; CD417 and CD2314, which are selective for
RAR
; CD437 (also named SR11248 or AHPN) and CD2325, which bind to
RAR
. These results are generally in good agreement with their
transactivational properties as reported previously (39, 40). Finally,
we used CD2624, which binds and activates all RXR isoforms, but not
RARs (36).
First, the efficacy of these retinoids in inducing growth arrest was
examined. Cells were treated for 5 days with different concentrations
of retinoids with or without CD2624 as indicated (Fig.
1), or CD2624 alone, to study the effects of single
activated RAR isoforms in the presence or absence of activated RXR,
respectively. After the last day, relative cell numbers were obtained
by means of a colorimetric assay, in which the absorbance at 490 nM directly correlates with the number of viable cells. The
absorbance values of control-treated cells was arbitrarily set at
100%, and all other values were normalized accordingly. Media and
retinoids were changed every 24 h to minimize loss of
concentration of intact ligands due to chemical decomposition, as well
as to minimize potential aberrant specificities by those byproducts
(41).
As shown in Fig. 1, all compounds cause growth arrest or a decrease in
viable cells in a dose-dependent fashion. The RAR and
RAR
compounds have an IC50 of about 10
7
M, and the RAR
and RXR compounds about 10
6
M. Human melanoma cells appear to have the same sensitivity
profile for some of these compounds (42). Simultaneous addition of RAR and RXR ligands renders the cells about 10-fold more sensitive, indicative of a synergistic rather than an additive effect. These results suggests that all RARs and RXR
and/or RXR
can, directly or indirectly, inhibit cellular proliferation.
Because RAR has been implicated in the
above described processes, we next studied the induction of the RAR
gene by two different methods. Transcription of this gene is rapidly
induced by RA (within 2 h), even in the absence of protein
synthesis (23, 24). Cells were transiently transfected with a plasmid,
pW1RAR
2pr-lucif, which contains the chromosomal fragment harboring
the RAR
2 promoter linked to the luciferase (Luc) gene (32).
Transcriptional activation of this promoter is then detected as
enzymatic luciferase activity in the cell extract (Fig.
2A). Three concentrations of each ligand were
chosen, varying between 10
8 and 10
5
M, based on their displayed efficacy in the cell
proliferation assay. Thirty-six hours after transfection, cells were
exposed to all ligands for an additional 12 h because this is the
earliest time point at which significant luciferase activity could be
detected (data not shown). Fig. 2A shows that the promoter
is activated by all ligands, including CD2624, in a
dose-dependent manner. Maximum induction levels are
generally reached between 6- and 15-fold at 10
7 and
10
6 M. These data correlate well with their
activity in the cell proliferation assay, and suggest that all
expressed receptors can activate this promoter.
The second method measures induction of the endogenous promoter. Cells
were treated for 8 h with a representative group of retinoids at a
concentration of 106 M for maximum induction.
In addition, cells were incubated at the same time in the absence or
presence of the protein synthesis inhibitor cycloheximide to test
whether induction is dependent on on-going protein synthesis. After
treatment, poly(A)+ RNA was isolated and separated by
agarose gel electrophoresis, and RAR
mRNA was detected by
Northern blot analysis (Fig. 2B). We again see that the
RAR
gene is rapidly induced by all tested agonists. Also, the level
of induction (10-15-fold) corresponds well with the values found in
the transfection assay. Thus, regulation of expression of this gene can
be mediated by ligands specific for all receptors, including RXR
and/or RXR
. The latter result confirms our previous report that RXR
can transcriptionally activate this promoter despite its unfavorable
(to RXRs)
RARE (28-30). Fig. 2B also shows that
cycloheximide does not abrogate transcription of this gene. This
suggests that all receptors, including RAR
whose mRNA levels are
very low (but still detectable) in the absence of added retinoids (Fig.
2B and Refs. 23-25), are likely already present in
sufficient numbers to initiate a functional response. Interestingly,
cycloheximide does not abrogate, but rather enhances induction by all
tested agonists in a strictly retinoid-dependent fashion;
on average, RNA levels increase to between 28- and 61-fold higher
levels as compared with control treated cells. Superinduction of the
RAR
promoter by cycloheximide was previously reported for treatment
with RA (23, 24) but our results show that isoform-specific ligands
also synergize with cycloheximide.
Thus, all ligands and, therefore, most likely all RARs as well as
RXR and/or RXR
can activate RAR
expression. Even though we
also observe a correlation between growth arrest and RAR
expression, our results show that the accumulation of RAR
occurs in the absence of RAR
-activating ligands. These data argue against a critical role
for ligand-bound RAR
in S91 cells.
After 5 days of treatment with the
RAR-specific ligands CD437 and CD2325 at 10
6
M, we observed a substantial loss in the number of adherent
cells (see below). In addition, compared with all other treated cells, the morphology of the remaining adherent cells is that of a
differentiated, melanocytic phenotype including dramatic cell
flattening and growth of dendritic extensions (Fig.
3A), which is independent of initial cell
density, although very sparsely seeded cells are more spindle-shaped than more densely growing cells (compare Fig. 3, B with
A, respectively). The RAR
-ligand-induced morphology
resembles that which is obtained after treatment with RA (Ref. 23, and
data not shown). These attached cells also display another marker for
melanocytic differentiation, increased intracellular melanin (23, 27)
(Fig. 3C). In contrast, the other ligands induce no or much less
melanin. This suggests that RAR
uniquely among the expressed
retinoid receptors mediates the differentiation of these melanoma
cells. Our results also imply that growth arrest may be a necessary but
not sufficient condition for morphological differentiation, indicative
of two distinct and separate pathways. A similar principle was
postulated for RA-dependent differentiation of
neuroblastoma (NB) cells (43).
Concomitant with Differentiation, RAR
Finally, we examined the mechanism(s) leading to
the loss of adherent cells upon addition of the RAR-specific ligands,
CD437 and CD2325, at 106 M. At this
concentration, substantial numbers of floating cells can be observed
after about 24 h treatment. These cells largely retain dye after
staining with trypan blue, suggesting that they are dead or dying (data
not shown). We reasoned that the two ligands could either be toxic to
the cells, which could lead to necrosis, or might induce genetically
programmed cell death (apoptosis). Retinoid-induced apoptosis has been
observed in a number of other retinoid-treated tumor cell lines
(44-57). To examine whether the latter mechanism would also apply
here, we tested for two different markers of apoptosis.
First, genomic DNA was isolated from all cells (adherent and floating)
that were treated for 24 h with our agonists, followed by analysis
by agarose gel electrophoresis. DNA laddering due to internucleosomal
fragmentation of DNA in multiples of 180 nucleotides, typical of
apoptotic cells, can be observed in the lanes with RAR
agonist-treated cells, but not in any of the other lanes (Fig.
4A).
We also applied a different method based on the sandwich enzyme immunoassay principle which detects and quantifies accumulating mono- and oligonucleosomes in the cytoplasmic fraction of apoptotic cells. Our results show that apoptosis occurs between 8 and 16 h of treatment with CD2325, after which it no longer increases (Fig. 4B). Consistent with the morphological observations and the amount of DNA laddering in Fig. 4A, similar final absorbance values in this assay are measured after treatment with CD437. In contrast, Am580, CD2314, and CD2624 show little or no increase in absorbance after 24 h (Fig. 4B), and RA does not cause significant apoptosis either.
Evidence That the Mechanism of RARTo initially
investigate whether the mechanism of apoptosis requires newly
synthesized proteins, we simultaneously incubated cells for 16 h
in the presence or absence of CD437 and CD2325, and with or without
cycloheximide. As shown in Fig. 5A, these agonists again induce extensive apoptosis as detected by our sandwich enzyme immunoassay. However, apoptosis can be completely blocked by the
addition of cycloheximide, whereas cycloheximide alone has no
significant effect on cell viability. This experiment shows that the
protein(s) which mediate apoptosis are not yet present in the untreated
melanoma cells, but are induced by the RAR agonists and suggest that
new gene transcription is required. Since functional RAR
is already
present in the melanoma cells, and CD437 and CD2325 are strong
activators of this receptor, it is quite conceivable that RAR
is
responsible for induction of these putative genes.
However, this could still be an indirect, rather than a direct
transcriptional effect. It is known for some time that liganded RARs
can interfere with AP-1 (Jun)-mediated gene transcription, possibly by
directly interfering with DNA binding by AP-1 through protein-protein
interactions. Conversely, AP-1 can also block DNA binding of RAR·RXR
heterodimers (58). It was recently reported that the anti-AP-1 activity
of liganded RARs, and not their transactivational properties, is
responsible for the growth-inhibitory effects of retinoids on certain
tumor cell lines (59-61). In our model, RA-liganded RARs could block
AP-1-mediated transcription, which could prevent induction of apoptosis
by AP-1. Possibly, CD437 (and CD2325) could bind to RAR in a
different fashion and alter its conformation such that it would no
longer interact with AP-1, and this release may then lead to
cycloheximide-sensitive apoptosis. To investigate this possibility, we
performed an EMSA, shown in Fig. 5B. As a source of
proteins, RAR
and RXR
were obtained by in vitro
translation in the rabbit reticulocyte system, whereas recombinant AP-1
(Jun) was commercially obtained. As probe, we used an oligonucleotide with the well established
RARE (25). As expected, unprogrammed and
RAR
- or RXR
-programmed lysate displayed little DNA binding activity (lanes 1, 9, and 10, respectively). When
RAR
and RXR
-programmed lysate are combined, DNA-binding
heterodimers are formed (lane 4). Binding is specific, as
this complex disappears in the presence of a 50-fold molar excess of
unlabeled
RARE oligonucleotide (lane 3), but not in the
presence of an equal excess of an oligonucleotide with an AP-1-binding
site (12-O-tetradecanoylphorbol-13-acetate-response element)
(lane 2). DNA-binding and mobility of RXR
·RAR
heterodimers is not significantly differentially affected by the
presence of 1 µM ligand RA or CD437 (lanes 5 and 7, respectively), which indicates that there are no
major differences in receptor conformation induced by these two
ligands. Next, to imitate the situation in our cells after addition of
ligands, we added pure Jun protein to the liganded heterodimer complex.
As seen in lane 6, we too observe that Jun completely
inhibits DNA-binding of RA-liganded RXR
·RAR
heterodimers as
their is no shifted complex visible anymore. The same observation is
made when Jun is added to CD437-bound RXR
·RAR
heterodimers. Thus, in vitro there is no discernable differential
ligand-specific effect in the behavior of liganded-RXR
·RAR
heterodimers with respect to their interaction with AP-1 (Jun). The
same appears to be true in vivo as well. If cells undergo
apoptosis because of unmasking of AP-1-mediated transcriptional
activity, it can be expected that direct activation of AP-1 activity
might lead to apoptosis. For this purpose, cells were treated for
16 h with increasing doses of phorbol 12-myristate 13-acetate
(also called 12-O-tetradecanoylphorbol-13-acetate). Phorbol
12-myristate 13-acetate is a well known activator of PKC and AP-1
activity. However, no apoptosis can be detected over this time frame in
the presence of up to 100 ng/ml (Fig. 5C). In the same
experiment, apoptosis starts to become measurable at 10
7
M CD437, and is fully established at our working
concentration of 10
6 M (Fig. 5C).
Similarly, there is also no effect in the presence of the PKA activator
8-Br-cAMP (Fig. 5C). These results all suggest that AP-1,
PKC, and PKA activities are not of critical importance for the
mechanism of CD437 and CD2325-induced apoptosis.
Finally, another mechanism could be at work here which would be
independent of RARs. It is possible that these retinoids induce oxidative stress due to the formation of reactive oxygen radicals, and
this may induce gene transcription. Transforming growth factor- has
been shown to induce apoptosis in certain cells through this mechanism (Ref. 62, and references therein). To investigate this
possibility, we incubated the cells for 16 h in the presence of
CD437 and increasing doses of up to 1 mM GSH. GSH is a free oxygen radical scavenger, and if this mechanism is applicable here it
might be expected that GSH could inhibit CD437-induced apoptosis.
However, as shown in Fig. 5C, addition of GSH has no measurable effect on CD437-induced apoptosis. This makes this mechanism
less probable here.
Retinoids are known to have profound effects on growth and
differentiation of cells in vivo and in vitro
(17-20). S91 melanoma cells provide a model system that allows the
study of neoplastic change of melanocytic cells into melanoma in a
controlled experimental setting. The entire process is strictly
retinoid-dependent, and, with the data presented here as well
as in other reports (23-25), all active and expressed RARs and RXRs in
S91 cells have now been determined. However, a list of all potential
players involved in the first step in the genetic cascade which leads
to growth arrest and differentiation does not reveal the importance of
each RXR and RAR isoform. This study attempts to dissect the individual roles of all receptors in these processes by using ligands that have
high specificity for certain isoforms only. In accordance with these
data, our results show that these agonists indeed have very specific
effects on cellular proliferation, viability, morphological differentiation, induction of RAR gene expression, and apoptosis, as
discussed below.
First, we show that inhibition of cell division and transcriptional
induction of the RAR gene can be mediated by all ligands, and thus,
most likely all RAR isoforms and RXR
and/or RXR
. Functional redundancy in RAR function has been observed before, both in
vivo and in vitro. For instance, RAR
and RAR
gene
knock-outs in transgenic mice do not cause the embryos to die in
utero, but they can develop relatively normally until birth
(63-65). RAR
knock-out mice have no discernable phenotype at all
(66). RAR
and RAR
knock-outs in F9 embryo carcinoma (EC) cells do
not abrogate RA-dependent induction of RAR
expression
(41, 67). Presumably, in all those cases as well as in others (68-71),
alternative receptors can substitute for functions performed by the
deleted RAR genes.
It is conceivable that RAR could play a major role in the RA-induced
differentiation of S91 cells (23). Indeed, exogenously introduced
RAR
can halt the growth of lung cancer cells (71), and RAR
gene
expression and/or RAR
mediated activity appears to be altered in
lung tumor cells (72-74) and other (pre)cancerous epithelial cells
(22, 75, 76). However, our results show that, in S91 cells,
ligand-bound RAR
is not required for growth arrest or
differentiation, because ligands that do not activate RAR
also cause
growth arrest. It is interesting that the RAR
ligands are about
equipotent in inducing RAR
gene transcription as the RAR
, RAR
,
and RXR ligands. In the absence of exogenously added retinoids,
expression of RAR
is extremely low. Thus, it appears that the
base-line level of RAR
in untreated cells is high enough to induce
transcription, which would also explain why inhibition of protein
synthesis by cycloheximide does not abrogate RAR
-mediated
transcription. It is unknown why cycloheximide enhances
retinoid-dependent transcription, but our results indicate that all RAR isoforms and RXR might interact with the same putative labile transcriptional repressor protein(s) (23).
Second, our results show that only the RAR-ligands induce
morphological differentiation toward the melanocytic phenotype which is
normally only seen after treatment with RA and 9-cis-RA (Ref. 23, and data not shown). This identifies RAR
as most likely to
be the critical RAR isoform which maintains these melanocytes in their
differentiated state, and prevents them from converting to melanoma. We
postulate that RAR
can uniquely modulate the expression of a set of
"differentiation" genes, which cannot be regulated by RAR
,
RAR
, or RXR. This conclusion is based on the assumption that the
selectivity of the ligands, which is determined in vitro at
4 °C, is preserved in vivo at 37 °C. This is difficult to determine directly due to the lack of mammalian cell lines which do
not express RARs and/or RXRs. However, the available evidence suggest
that ligand-binding selectivity is indeed maintained over this
temperature range (38, 77).
RAR-induced differentiation may be mediated by a novel type of
RAR
-responsive RARE, present in genes which are activated or
repressed by only this particular receptor, or could involve new
isoform-specific cofactors similar in function to those in the AP-1
family (78). Differential activation of certain RAREs by different
receptor isoforms has been shown before (Ref. 33, and see below).
Previously, a unique role for RAR among RARs in the differentiation
of tumor cells was established in the more "primitive" embryo
carcinoma (EC) cell lines, NTera2 and F9, which differentiate into a
neuronal-looking, phenotype-type and parietal endoderm after
administration of RA, respectively (41, 79). Separate transfection of
RAR
, RAR
, or RAR
expression vectors into NTera2 cells showed
that only the latter transfectant gives rise to a cell with a
differentiated phenotype. Somewhat puzzling, however, is that this
RAR
-expressing transfectant will now differentiate in the absence of
exogenously added RA (80). Gene ablation experiments in F9 EC cells
showed that RAR
is mostly responsible for differentiation. This
laboratory also showed that different RAR isoform knock-outs differentially affect the expression of RA-regulated genes in F9 cells
(41, 67). However, reintroduction of plasmids overexpressing either
RAR
or RAR
(but not RAR
) in RAR
/
cells showed that both RAR isoforms could restore differentiation (25). Consistent with
our data, as well as that of others, they suggested that certain
genetic functions are redundant (63-65, 68, 69, 81) but others may be
regulated by specific RAR isoforms (33, 67, 82-84), perhaps through
the aforementioned isoform-responsive RAREs. A unique role for RAR
in neuroblastoma cells is less well established than that in EC cells.
Marshall et al. (43) showed that RAR
expression is
significantly higher in primary neuroblastoma tumor tissue compared
with advanced or disseminated neuroblastoma tumors. Also, transfection
of an RAR
expression vector into the neuroblastoma cell line BE(2)-C
altered its neuritic differentiation potential. However, other reports
suggest that RAR
and/or RAR
is involved in differentiation of
neuroblastoma cells (85-87).
Finally, we observed that the RAR ligands, CD437 and CD2325, not
only induced differentiation, but also apoptosis. Retinoids have been
shown to induce apoptosis in several other different tumor cell lines,
and can occur during or after differentiation, or occur in the absence
of any differentiation (44-57). Recently, induction of growth arrest
and apoptosis by CD437 (or AHPN) was reported in the breast cancer
lines MCF-7 and MDA-MB-231, which appears to take place with similar
kinetics as in our cells (56). This mechanism(s) is independent of p53,
bax, and bcl-2, and may be dependent on regulation of WAF/CIP1. In
addition, the authors suggest that it is at least partly independent of
RARs because promyelocytic HL-60R cells, a RA-resistant subclone of
HL-60 cells, are also growth arrested by CD437 (56), although the
authors do not report whether HL-60R cells will also undergo apoptosis after treatment with CD437. On the other hand, a report by Liu et
al. (50) showed the involvement of RAR
in retinoid-mediated apoptosis in breast cancer cells. It is clear that one should be
cautious in extrapolating results from one cell line to another. For
instance, with regard to HL-60 cells, apoptosis is mediated by RXRs
(53) whereas RAR
mediates their differentiation (68). According to
our data, these receptors do not appear to be very important in the S91
melanoma model. Another example is RA-dependent apoptosis
in the tracheobronchial cell line SPOC-1. Here, RAR
agonists cause
apoptosis but CD437 has no effect (57). It appears that the cell type
strongly influences which pathway leading to differentiation and/or
apoptosis is activated.
Our evidence suggests that in S91 cells RAR is activated by CD437
and CD2325, and our experiments with cycloheximide suggest that
transcription and synthesis of new protein(s) which mediate apoptosis
needs to take place. This would require some time to develop, and our
observation that apoptosis becomes apparent about 8 h after
administration of agonist would certainly not be inconsistent with this
hypothesis. Moreover, we found little or no evidence which would
support alternative, unexplained receptor-independent mechanisms,
although we cannot completely rule these out.
Activated RARs can interfere with AP-1-mediated transcription, and we
investigated the possibility whether the RAR agonists would alter
the conformation of the receptor in a different way than RA. We
speculated that RA-bound receptors might block AP-1 activity and
subsequent apoptosis, whereas the RAR
agonist-bound receptors may
not. This could explain why RA does not cause apoptosis in contrast to
the RAR
agonists. However, as judged by EMSA, both types of ligands
allow the activated receptor to interact with AP-1. We were also unable
to directly induce apoptosis by activation of AP-1 and PKC by phorbol
12-myristate 13-acetate (and PKA by 8-Br-cAMP). In addition, it was
reported that increased activity of AP-1 takes place during
cAMP-induced melanogenesis in the related B16 cells (88). These results
all argue against involvement of AP-1 during apoptosis. Another
possibility could be that CD437 and CD2325, and none of the other
retinoids tested here, could lead to oxidative stress due to the
formation of reactive oxygen intermediates. This could also lead to
induction of gene transcription, and transforming growth factor-
is
known to cause apoptosis through this mechanism (62). However, we found
that GSH, a well established oxygen radical scavenger, does not block CD437-induced apoptosis. Again, these results certainly do not favor
this mechanism, and make it less likely than a receptor-mediated mechanism. In addition, the observed requirement for new protein synthesis argues against an interleukin 1
-converting enzyme-type mediated mechanism, as these proteins are ubiquitously present in the
cytoplasm.
One important question which remains to be answered is why RA induces
differentiation, but not apoptosis, in contrast to the synthetic RAR
agonists, even though they both must activate RAR
. It is not due to
an intrinsic inability of RA to cause cell death: in two other reports
of concomitant apoptosis and differentiation by retinoids, one in a
particular subclone of F9 EC cells (45), and the other in P39
myelomonocytic leukemia cells (44), the effects are induced by RA,
albeit with slower kinetics and in a relatively small number of cells.
We hypothesize that, under certain conditions, the RAR
agonists can
be stronger activators of RAR
than RA, perhaps mediated by new
cofactors (78). We speculate that there may be genes which have an RARE
whose degree of transcriptional activation is dependent on the type of
agonist bound to the receptor. In case of CD437 and CD2325 this may
then lead to apoptosis. It has been shown before that conformationally restricted agonists can exhibit gene selectivity in the context of a
given RARE, which could have important physiological consequences (89).
For instance, CD437 and CD2325 are more powerful inducers of F9 EC cell
differentiation than RA (38).
The unraveling of the complex retinoid-mediated pathways in the S91
melanoma model will be important for our understanding of neoplastic
transformation, and this work is currently in progress in our
laboratory. In this report, we identified RAR as the responsible RAR
isoform in S91 cells which operates a genetic switch of the molecular
processes causing the malignant transformation of melanocytes into
melanoma cells, and probably apoptosis. Hopefully, this may eventually
lead to the design of more effective treatments and/or diagnostics
in vivo.
We thank Adam Lipson for technical assistance and Dr. Paul Yen for critically reading the manuscript. We are also greatly indebted to Drs. Uwe Reichert and Serge Michel for help throughout this work.