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Int. J. Cancer: 71, 497?504 (1997)
r 1997 Wiley-Liss, Inc.
Publication of the International Union Against Cancer
Publication de l?Union Internationale Contre le Cancer
ACTIVITY OF RETINOIC ACID RECEPTOR-g SELECTIVELY BINDING
RETINOIDS ALONE AND IN COMBINATION WITH INTERFERON-g
IN BREAST CANCER CELL LINES
Martin WIDSCHWENDTER1*, Gu?nter DAXENBICHLER1, Zoran CULIG2, Serge MICHEL3, Alain G. ZEIMET1, Manfred G. MO?RTL4,
Andreas WIDSCHWENDTER5 and Christian MARTH1
1Department of Obstetrics and Gynecology, University of Innsbruck, Innsbruck, Austria
2Department of Urology, University of Innsbruck, Innsbruck, Austria
3CIRD Galderma, Sophia Antipolis, France
4Department of Anesthesia and Intensive Care, University of Innsbruck, Innsbruck, Austria
5Department of Medical Biochemistry, University of Innsbruck, Innsbruck, Austria
Retinoids modulate several cell functions and especially
inhibit the growth of a wide variety of cells including breast
cancer. Retinoic acid receptor-g (RAR-g) has been shown to
mediate the antiproliferative activity of retinoids. To further
test this hypothesis we examined the effects of different
RAR-g selectively binding retinoids (CD2325, CD2247, CD666
and CD437) on breast cancer cell lines. With exception of
CD2247, all retinoids inhibited proliferation of MCF-7, SKBR-3,
T47D and ZR-75-1 breast cancer cell lines, similar to the
natural compound all-trans retinoic acid (ATRA). In addition,
all 4 compounds were able to act synergistically with interferon-g (IFN-g) in all breast cancer cell lines including the
retinoid-resistant BT-20 and 734-B lines. In functional transactivation assays we demonstrated that only in the MCF-7 cell
line, TPA-mediated AP-1 activity was suppressed only by
ATRA and CD2325, whereas in SKBR-3, another RA-sensitive
breast cancer cell line, it was not. The synergistic antiproliferative activity involving retinoids and IFN-g could not be
explained by an enhanced anti-AP-1 activity. No correlation
was found between expression of RARs and cellular retinoic
acid binding proteins (CRABPs) and antiproliferative effects
of the retinoids. RAR-g selectively binding retinoids are
potent inhibitors of breast cancer cell proliferation, alone and
in combination with IFN-g. For this reason and because of a
possible low toxicity, as compared with retinoic acid, we
speculate that these RAR-g selective binding retinoids might
be of clinical importance. Int. J. Cancer 71:497?504, 1997.
r 1997 Wiley-Liss, Inc.
Retinoids are known to possess antiproliferative, differentiative
and immunmodulatory properties. The key molecules are the
binding proteins CRABP I and II (cellular retinoic acid-binding
protein), the retinoid receptors (RAR-a, RAR-b and RAR-g) and
retinoid X receptors (RXR-a, RXR-b and RXR-g), which are part
of the steroid/thyroid hormone receptor superfamily (Sporn et al.,
1994). A growing body of evidence derived from clinical research
supports the concept that retinoids are useful substances in the
prevention and treatment of cancer. Retinoids partly alone, partly in
combination with biological response modifiers or chemotherapy
have proved to be effective in skin diseases, acute promyelocytic
leukemia, cervical cancer and other malignancies (Sporn et al.,
1994). When combined with tamoxifen and interferon (IFN),
retinoids induce remission in heavily pretreated patients with
metastatic breast tumors (Recchia et al., 1995). The pleiotropic
biological activities of natural retinoids and related compounds,
however, produce a number of undesirable side effects, which limit
their clinical applications. In breast cancer cell lines, retinoids
alone or in combination with interferons exhibit strong antiproliferative activity (Marth et al., 1986, 1993; Widschwendter et al.,
1996). We have shown that IFN-g-mediated increase in RAR-g and
suppression of retinoic acid (RA)-mediated CRABP II activation
may play a role in synergistic inhibition of proliferation in breast
cancer cell lines (Widschwendter et al., 1995). Extending these
findings, we compared the antiproliferative effects of RAR-g
selectively binding retinoids CD2325, CD2247, CD666 and CD437
on several breast cancer cell lines with those of natural retinoids,
alone and in combination with IFN-g. Fanjul et al. (1994) have
shown that retinoids may inhibit proliferation by reducing AP-1
activity. Therefore, we were interested in checking whether these
retinoids were also able to reduce AP-1 activity. To compare levels
of RARs and CRABPs with response to these retinoids we
performed Northern blots. Modulation of CRABP is a possible
mechanism influencing retinoid action. As a consequence of the
previous finding that all-trans retinoic acid (ATRA) induces
CRABP II, we were also interested in CRABP II promoter
activation by CD2325.
MATERIAL AND METHODS
Reagents
CD2325, CD2247, CD666 and CD437 (Fig. 1) were kindly
provided by Dr. U. Reichert (CIRD Galderma, Sophia Antipolis,
France), and ATRA by Dr. W. Bollag (Hoffmann-La Roche, Basel,
Switzerland). For all experiments, 1-mM solutions were prepared
in DMSO and further diluted in complete culture medium. To avoid
degradation and isomerization of the retinoids, only fresh solutions
were used. The human recombinant DNA-derived IFN-g was
kindly provided by Dr. G. Adolf (E. Boehringer Institute, Vienna,
Austria). The preparation was essentially pure, and exerted an
antiviral activity of 2 3 107 U/mg protein. IFN-g was diluted in
minimum essential medium (MEM) containing 15% fetal bovine
serum (FBS) and stored at 270蚓.
Cell culture
The MCF-7, BT-20, SKBR-3, 734-B, ZR-75-1, T47D and
Hs578T human breast cancer cell lines were cultured as described
by Marth et al. (1993). Briefly, the cells were maintained in MEM
containing 10% FBS (both from Eurobio, Paris, France); they were
seeded into 24-well tissue culture plates (Nunc 146485, Roskilde,
Denmark) in complete culture medium for evaluation of proliferation. After treatment with ATRA or CD2325 alone or in combination with IFN-g or the vehicle alone, cells were detached with the
help of trypsin (0.05%)-EDTA (0.02%) in Dulbecco?s phosphatebuffered saline (PBS, Biological Industries, Kibbutz Beth Haemek,
Israel), and their number was assessed using an electronic particle
counter (Coulter, Dunstable, UK) as described by Marth et al.
(1993).
Northern blot analysis
Northern blot analysis was done as described by Widschwendter
et al. (1995). Briefly, total cellular RNA was extracted by the
Contract grant sponsor: Jubila?umsfonds der O?sterreichischen Nationalbank, 5788.
*Correspondence to: Department of Obstetrics and Gynecology, University of Innsbruck, Anichstrasse 35, A-6020 Innsbruck, Austria. Fax: 0043
512 504 3112.
Received 16 December 1996
498
WIDSCHWENDTER ET AL.
FIGURE 1 ? Structure, name and KD (nM)-values for RAR-a, RAR-b and RAR-g for RAR-g selectively binding retinoids CD2325, CD2247,
CD666 and CD437.
guanidine thiocyanate method. Ten micrograms of total RNA
mixed with ethidium bromide were run on denaturing 1% agaroseformaldehyde gels and transferred to nylon membranes (Stratagen
Flash Nylon Membranes, La Jolla, CA) by Northern blotting. The
sheet thus prepared was fixed and photographed under ultraviolet
(UV) light (to demonstrate comparable RNA levels) and hybridized
with digoxigenin-labelled DNA probes as previously described.
Detection of digoxigenin-labelled nucleic acids by chemiluminescence enzyme immunoassay on nylon membranes was carried out
following the manufacturer?s instructions (DIG Luminescent Detection Kit, Boehringer Mannheim, Vienna, Austria). Filters were
exposed to autoradiographic films (Hyperfilm, Amersham, Solna,
Sweden) for 5 hr.
Statistics
Differences in median cell number were analyzed with the
Wilcoxon U-test. Interaction of CD2325 with IFN-g was evaluated
according to the recommendations of Berenbaum (1981) and Chou
and Talalay (1984), using the computer program Dose-Effect
Analysis With Microcomputer (Elsevier Biosoftware, Cambridge,
UK). Dose-effect curves were plotted with either agent alone and
with their combination. From these experiments a combination
index (CI) was calculated to characterize the quality of interaction:
CI 5 1 indicates an additive interaction, whereas CI . 1 represents
antagonistic and CI , 1 synergistic activity. This type of calculation is very similar to isobolarogram construction and has the
advantage of preventing errors caused by dose-response curves
which are not simply exponential. A most widely used model for
analyzing interaction of substances is the effect-multiplication
criterion. Several sources of error may, however, occur, and
therefore experiments with various dose combinations and evaluations of effects by appropriate mathematical methods are proposed.
CAT assays
MCF-7 cells were grown on 24-well plates. The CAT reporter
plasmid 5 3 TRE TATA CAT (Jonat et al., 1990) was kindly
provided by Dr. A.C.B. Cato (Karlsruhe, Germany), and the CAT
reporter CRABPII-RARE2 (Durand et al., 1992) by Dr. P. Chambon (Strasbourg, France). Liposome-mediated transfection was
performed under serum-free conditions. The DNA solution was
RAR-g SELECTIVE BINDING RETINOIDS AND IFN-g IN BREAST CANCER CELLS
mixed with lipofectamine transfection reagent (GIBCO BRL,
Paisley, UK; 3 ml/mg plasmid DNA) and added to the cells. After
an incubation period of about 10 hr, medium was changed to
phenol red-free medium supplemented with 1% charcoal-treated
serum, and substances were added. Twenty-four hours after supplementation, the medium was removed and the cells were frozen.
CAT activity was assessed with a modified mixed-phase assay: the
cells were incubated with CAT mix [1.3 mg/ml chloramphenicol,
0.25 湣i 3H-labeled (NEN, Boston, MA) and 60 nM unlabeled
acetyl-coenzyme A (Sigma, St Louis, MO)] in 0.3 M Tris HCl, pH
7.0, at 55蚓 for up to 2 hr. A scintillation cocktail insoluble in water
(Optiscint, Pharmacia, Uppsala, Sweden; 0.75 ml) was added to the
samples, and acetyl chloramphenicol was measured with a b-scintillation counter.
RESULTS
Effect of ATRA and RAR-g selective binding retinoids on breast
cancer cell proliferation
We analyzed the effects of ATRA and CD2325, CD2247, CD666
and CD437 on the proliferation of MCF-7, ZR-75-1, T47D,
SKBR-3, 734-B and BT-20 breast cancer cell lines (Fig. 2). Under
our culturing conditions, ATRA significantly inhibited proliferation
of MCF-7, ZR-75-1, T47D and SKBR-3 cell lines but showed no
effect on 734-B and BT-20 cells. CD2325 and CD666 exhibited our
antiproliferative activity similar to that of ATRA. CD437, mainly at
concentrations of 1027?1026 M, demonstrating a strong antiproliferative potency on the RA-sensitive as well as RA-refractory cell
lines. On the other hand, the CD2247 compound inhibited proliferation only in MCF-7 and T47D cells.
Effects of RAR-g selective binding retinoids in combination
with IFN-g on proliferation of breast cancer cell lines
In our previous studies we were able to demonstrate a synergistic
effect of RA with IFNs with regard to proliferation inhibition. To
further substantiate the observation that RAR-g might be strongly
involved in proliferation inhibition, we tested the RAR-g selectively binding retinoids CD2325, CD2247, CD666 and CD437 in
combination with IFN-g. In order to analyze this drug interaction,
we performed isobolograms and calculated the CI values (Table I).
CI values ,1 are indicative of synergistic interaction. CD2325,
CD2247, CD666 and CD437 in combination with IFN-g demonstrated a strong proliferation inhibition on MCF-7, SKBR-3 and
even in the RA-resistant cell line BT-20. The synergistic effect was
lowest with the combination of CD437 and IFN-g. In 3 other cell
lines, ZR-75-1, T47D and 734B, treatment with CD2325 and
IFN-g also led to synergistic proliferation inhibition.
Anti-AP-1 activity of RAR-g selective binding retinoids alone
and in combination with IFN-g
Since antiproliferative action of retinoids may mainly be due to
anti-AP-1 activity (Fanjul et al., 1994) we tested whether the
RAR-g selectively binding retinoids were able to suppress AP-1
promoter activity and whether IFN-g was able to enhance an
anti-AP-1 effect. As shown in Figure 3 (4 independent experiments), on MCF-7 cells, TPA-mediated AP-1 activity was suppressed by IFN-g or ATRA to 75% vs. 57%, respectively. Among
the RAR-g selectively binding retinoids, only the CD2325 compound affected AP-1 activity. Only in combination with CD666 did
IFN-g exert a significant additional anti-AP-1 effect. To further
evaluate the effects of CD2325 on AP-1 activity, we tested 2
RA-sensitive and 2 RA-resistent cell lines. Figure 4 (3?4 independent experiments) shows that only in the 2 RA-sensitive cell lines
MCF-7 and SKBR-3 did TPA activate the AP-1 promoter. Constitutive values of AP-1 activity were comparable in all 4 cell lines used
(data not shown). In the 2 RA-resistant cell lines BT-20 and
Hs578-T, TPA was not able to activate AP-1. Only on MCF-7 cells,
RA as well as CD2325 suppressed TPA-mediated AP-1 activity.
Interestingly, in SKBR-3, an estrogen receptor-negative (tested,
499
results not shown) but RA-sensitive cell line, neither ATRA nor
CD2325 suppressed AP-1 activity.
Effects of retinoids and IFN-g on CRABP II promoter activity
In our previous report we stated that synergism of retinoids and
IFNs may partly be due to IFN-mediated suppression of retinoidinduced expression of CRABP II. Figure 5 shows the results of 6
independent experiments. CD2325 was at least as potent as ATRA
on CRABP II promoter activation. IFN-g only slightly suppressed
the CD2325-mediated effect, but reduced ATRA-mediated CRABP
II activation to 50%. Northern blots also showed CRABP II
upregulation by CD2325; this effect was not suppressed by IFN-g
(data not shown).
Expression of RAR- and CRABP-mRNA in breast cancer cell lines
To correlate growth effects on breast cancer cell lines and RAR
or CRABP effects, we performed Nothern blots (Fig. 6). RAR-b
and CRABP I were not expressed in the breast cancer cell lines we
examined. The highest expression of RAR-a was found with
MCF-7 cell lines, whereas in T47D, SKBR-3, BT-20 and 734B a
comparable lower amount of this transcript was detected. Hs578T
was negative for RAR-a. The ranking of RAR-g expression was as
follows: MCF-7 5 T47D 5 734B , BT-20 5 Hs578T , SKBR-3.
CRABP II transcripts were expressed at a very high extent in
MCF-7 and T47D cells, at a lower extent in SKBR-3, BT-20 and
734B and were undetectable in the Hs578T cell lines. Comparing
the growth data with RAR and CRABP expression, no correlation
could be found. For instance, MCF-7 and T47D cells exhibited
comparable expression patterns, while T47D cells were more
RA-sensitive. Comparing the highly RA-sensitive SKBR-3 with
the RA-resistant 734B cell line, we observed comparable expression patterns, except for a higher RAR-g value in 734B cells.
DISCUSSION
Combinations of retinoids with IFNs have been reported to be
very potent inhibitors of breast cancer cell proliferation (Marth et
al., 1986, 1993; Widschwendter et al., 1996). We studied the
mechanisms of this synergistic action and found that IFN-g
induced expression of RAR-g and suppressed RA-mediated induction of CRABP II, a molecule which may be responsible for
sequestration of RA, thus limiting its distribution and biological
effects (Widschwendter et al., 1995). Additional evidence for an
involvement of RAR-g in retinoid signalling comes from studies
with a retinoid-resistant human teratocarcinoma cell line: transfection with a vector overexpressing RAR-g restored ATRA, and
induced growth inhibition and differentiation (Moasser et al.,
1994). Murine F9 teratocarcinoma cells, which have lost RAR-g
expression through targeted gene disruption, do not exhibit the
morphologic differentiating effects of ATRA (Boylan et al., 1993).
Overexpression of RAR-g in neuroblastoma cells suppresses the
malignant phenotype and alters the differentiation potential (Marshall et al., 1995). The present study extends our analyses to the
RAR-g selective binding retinoids CD2325, CD2247, CD666 and
CD437. All these retinoids demonstrated a high selectivity for
binding to RAR-g. The potency of activation of different RARs has
been correlated well with binding data in the case of CD437 and
CD666 (Bernard et al., 1992). Despite high selectivity for binding
to RAR-g, the AC50 value (the retinoid concentration producing
half maximal activation) of CD2325 for RAR-g is only 4?6-fold
higher than for the 2 other RARs.
Our results demonstrate that with the exception of CD2247, all
RAR-g selectively binding retinoids effectively inhibit growth of
breast cancer cell lines as potently as RA. These retinoids also
inhibit proliferation of human melanoma cells (Schadendorf et al.,
1994). Correlating the retinoid binding affinity to RARs with
retinoid inhibition of growth of ER1 MCF-7 cells, Dawson et al.
(1995) observed that RAR-a is the retinoid receptor involved in the
inhibition of adherent cell growth. This appears to not contradict
our results, since Taneja et al. (1995) demonstrated the possibility
500
WIDSCHWENDTER ET AL.
FIGURE 2 ? Effects of retinoic acid and RAR-g selectively binding retinoids on proliferation of breast cancer cell lines. Cell lines were treated
with all-trans retinoic acid (?), CD2325 (s), CD2247 (d), CD666 (j) or CD437 (h) in concentrations of 10210?1026 M as indicated on the
abscissa. Results are presented as mean number of 6 wells counted in comparison with untreated controls (5100%). The coefficient of variation
was always below 10%. For reasons of clarity it is not shown.
of a functional redundancy between the various RARs: they
reexpressed RAR-g or overexpressed RAR-a in RAR-g-null F9
cells and were able to restore both target-gene activation and
differentiation potential. Moreover, the RAR-a-selective antagonist Ro 41-5253 inhibits SKBR-3 cell proliferation alone, but
diminishes all-trans-retinoic acid-mediated growth effects while
RAR-g SELECTIVE BINDING RETINOIDS AND IFN-g IN BREAST CANCER CELLS
TABLE I ? EFFECTS ON PROLIFERATION OF COMBINED TREATMENT
OF BREAST CANCER CELL LINES WITH RAR-g SELECTIVELY BINDING
RETINOIDS (CD2325, CD2247, CD666 AND CD437) AND IFN-g,
INDICATED AS A COMBINATION INDEX (CI)1
MCF-7
SKBR-3
BT-20
ZR-75-1
T47 D
734-B
CD2325
CD2247
CD666
CD437
CD2325
CD2247
CD666
CD437
CD2325
CD2247
CD666
CD437
CD2325
CD2325
CD2325
IC50
IC75
IC90
IC95
0.25
0.28
0.01
0.37
0.55
0.29
0.13
0.75
0.59
0.29
0.35
0.93
0.24
0.40
0.89
0.10
0.45
0.01
0.49
0.49
0.36
0.08
0.81
0.54
0.36
0.28
0.88
0.29
0.37
0.65
0.06
0.73
0.01
0.43
0.43
0.47
0.05
0.91
0.57
0.47
0.29
0.86
0.51
0.36
0.49
0.04
0.78
0.01
0.75
0.40
0.58
0.04
0.92
0.61
0.57
0.28
0.85
0.76
0.36
0.41
1CI was calculated on the multiple drug effect equation derived by
Chou and Talalay (1984), using computer software. CI , 1, 5 1 and
.1 indicate synergism, additive effect and antagonism, respectively. CI
values are shown for different inhibitory concentrations (IC).
501
not affecting synergistic interaction of all-trans-retinoic acid plus
IFN-g (Marth et al., 1993). Various results concerning expression
of RAR-b have been published (Liu et al., 1996, and references
therein). We did not detect RAR-b transcripts in control or
RA-treated cells (Widschwendter et al., 1995, 1996; Fig. 6),
indicating that this receptor does not play a role in inhibiting
proliferation in our cell culture system.
Fanjul et al. (1994) state that the antiproliferative activity of
retinoids may be mediated by their anti-AP-1 activity. To test this
hypothesis in our system we performed transfection assays and
found that AP-1 activity was stimulated only in RA-sensitive cell
lines, and only by TPA. Similar data were published by van der
Burg et al. (1995). In contrast to their results, we did not find high
constitutive activity of AP-1 in hormone-insensitive cell lines. The
SKBR-3 cell line, which is known to be estrogen receptor-negative,
exhibits TPA-inducible AP-1 activity. AP-1 activity was suppressed
only in the MCF-7 breast cancer cell line by ATRA and CD2325.
The other cell lines were refractory to retinoid-mediated AP-1
suppression. IFN-g exhibited modest anti-AP-1 activity. Only in
combination with CD666 did IFN-g have a significant additional
anti-AP-1 effect. CD437 exerts its antiproliferative effects in an
AP-1-independent manner (Shao et al., 1995), CD437 inducing
G0/G1 arrest and apoptosis via a unique pathway which appears to
FIGURE 3 ? Regulation of TRE-dependent transcriptional activity in MCF-7 breast cancer cells. MCF-7 cells were transfected with the
TRE-tk-CAT reporter plasmid and subsequently treated with vehicle only or cultured in the presence of TPA (100 ng/ml) alone or in combination
with IFN-g (10 ng/ml), ATRA (1 然/l) or the RAR-g selectively binding retinoids (CD2325, CD2247, CD666 and CD437) (1 然/l) alone or in
combination with IFN-g (10 ng/ml) for 24 hr. CAT activity determined in untreated cells was subtracted from activity in treated cells. Bars present
the mean of chloramphenicol conversion 6 SEM in TPA 6 IFN-g/retinoid-treated cells compared with TPA-treated controls (5100%). At least 4
independent experiments were done in triplicate.
502
WIDSCHWENDTER ET AL.
FIGURE 4 ? Regulation of TRE-dependent transcriptional activity in
different breast cancer cells. The RA-sensitive MCF-7 and SKBR-3
and the RA-resistant BT-20 and Hs578T cells were transfected with the
TRE-tk-CAT reporter plasmid and subsequently treated with vehicle
only (open bars), TPA (100 ng/ml) (solid bars) alone or in combination
with ATRA (1 然/L) (hatched bars) or the RAR-g selectively binding
retinoid CD2325 (gray bars) for 24 hr. The mean of the conversion of
chloramphenicol as measured in at least 3 independent experiments is
shown (6SEM), relative to that in nontreated controls (set at one).
FIGURE 5 ? Regulation of CRABP II-RARE2-dependent transcriptional activity in MCF-7 breast cancer cells. MCF-7 cells were treated
with ATRA (1 然/l) or with CD2325 (1 然/l) alone or in combination
with IFN-g (10 ng/ml) or with ATRA (1 然/l) and IFN-g (10 ng/ml) for
24 hr. CAT activity in untreated cells was subtracted from activity in
treated cells. Results are presented as the mean chloramphenicol
conversion 6 SEM in treated cells in relation to untreated controls
(5100%). At least 6 independent experiments were performed in
triplicate.
involve activation of known downstream effectors of p53 in a
p53-independent manner.
Treatment of MCF-7 cells with the potent AP-1 activator TPA
blocks cell proliferation (Kennedy et al., 1992). In contrast,
FIGURE 6 ? Expression of mRNA for RAR-a, RAR-b, RAR-g and
CRABP I and II in different breast cancer cell lines. Northern blot
analysis was performed on 10 痢 of total RNA as described in Material
and Methods. The data are representative of 2 separate experiments.
Staining of total RNA with ethidium bromide confirmed the integrity of
RNA and showed comparable RNA loading in each lane (photographs
not shown for reasons of clarity).
retinoids that selectively inhibit AP-1 activity and do not activate
transcription also inhibit proliferation (Fanjul et al., 1994). We
hypothesize that the 2 major receptor activities, transcriptional
activation and anti-AP-1 activity, require 2 different receptor
configurations induced or stabilized by the ligand. ATRA, with its
flexible polyene side chain, is apparently able to accommodate both
receptor configurations (Delescluse et al., 1991). The conformationally restricted retinoids identified by Fanjul et al. (1994) favor the
anti-AP-1 configuration. These retinoids fail to inhibit the proliferation of F9 cells and do not induce differentiation, whereas in the
T47-D cell line they reduce proliferation as determined by cell
counting, but are not as potent as ATRA. The results of Boylan et
al. (1993) indicate that RAR-g is involved in the regulation of
differentiation-specific genes. Therefore, induction of the antiAP-1 pathway appears to be not exclusively responsible for
proliferation inhibition of breast cancer cell lines by retinoic acid.
The antiproliferative effects of the RAR-g selectively binding
retinoids can be significantly enhanced by IFN-g. IFN-g appears to
be able to ??sensitize?? primary retinoid-insensitive cells, namely
BT-20 and 734-B, for retinoid action. On the other hand, treatment
of MCF-7 cells with the combination of ATRA or RAR-g
selectively binding cells and IFN-g did not further enhance
anti-AP-1 activity, meaning that there must be an additional factor
which regulates proliferation. It is very interesting that SKBR-3, an
estrogen receptor-negative breast cancer cell line, in contrast to the
other estrogen-independent cell lines (BT-20, 734B), is very
sensitive to retinoid-mediated antiproliferative effects. In contrast
to MCF-7 cells in the SKBR-3 breast cancer cell line, TPAmediated AP-1 activation was neither suppressed by ATRA nor by
CD2325. We looked for differences between SKBR-3 and other
estrogen receptor-negative cell lines and found that the ras
protooncogene-related protein rhoB is strongly expressed in
SKBR-3 but is absent in all other estrogen receptor-negative cell
lines examined (de Cremoux et al., 1994). Retinoic acid-mediated
regulation of this pathway is the subject of investigations in our
laboratory.
In our previous work we indicated that IFN-g-mediated suppression of RA-induced CRABP II expression may be partly involved
in the synergistic antiproliferative action of the combination of
these 2 substances. The induction of CRABP II transcription is
RAR-g SELECTIVE BINDING RETINOIDS AND IFN-g IN BREAST CANCER CELLS
mediated by RAR-RXR heterodimers bound to DR1- and DR2repeated motifs (Durand et al., 1992). To test the activity of
retinoids alone and in combination with IFN-g, we used a
DR1-specific reporter plasmid. CD2325 is as potent as ATRA in
activating this promoter. The combination of CD2325 and IFN-g
only slightly reduces promoter activity, whereas ATRA-mediated
activation is reduced to half the maximal values by IFN-g. We
therefore hypothesize that activation of the CRABP II gene may be
regulated by various pathways depending on the retinoid used, and
that DR1 activation may be blocked by IFN-g.
Our Northern blot analysis results reveal no correlation between
the expression of the RAR- and CRABP-untreated breast cancer
cell lines and the response to ATRA or the RAR-g selectively
binding retinoids. MCF-7 cells exhibited a relatively high expression of RAR-a and RAR-g compared with SKBR-3; on the other
hand, the SKBR-3 cell line is more sensitive to ATRA- and RAR-g
selectively binding retinoid-mediated proliferation inhibition than
the MCF-7 cell line. Therefore, we conclude that RAR and CRABP
expression in untreated breast cancer cell lines is not a good
predictor of RA-sensitivity.
Finally, we cannot rule out the possibility that RAR-g selectively
binding retinoids are taken up to a greater extent than ATRA, and/or
are metabolized to a lesser extent, in comparison with the
effectiveness of RAR-g selectively binding retinoids vs. ATRA.
503
Our data indicate that RAR-g selectively binding retinoids can
be potent antiproliferative agents on breast cancer cell lines, either
alone or in combination with IFN-g. Their antiproliferative potency
can be explained only in part by anti-AP-1 activity; at least in part,
other mechanisms (e.g., activation of differentiation-specific genes
or modulation of other growth-inducing factors or receptors)
appear to be responsible for growth inhibition. In conclusion, we
speculate that these compounds might have clinical importance
because of their possibly low toxicity compared with ATRA and
other synthetic and natural (e.g., 9-cis RA) retinoids and because of
their synergistic interaction with IFN-g.
ACKNOWLEDGEMENTS
We thank Ms. I. Gaugg for excellent technical assistance, and Dr.
E. Preuss for layout.
NOTE ADDED IN PROOF
After this article was submitted for publication, Fanjul et al.
(1996) also reported a potential role for RAR-g in inhibiting breast
cancer cells by means of selective retinoids and IFNs.
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