-
[CANCER RESEARCH 51. 4804-4809. September 15. 1991]
Identification of Retinoids with Nuclear Receptor
Subtype-selective Activities1
JürgenM. Lehmann, Marcia I. Dawson, Peter D. Hobbs, Matthias
Husmann, and Magnus Pfahl2
Cancer Center, La Jolla Cancer Research Foundation, La Jolla,
California 92037 [J. M. L., M. H., M. P.], and Life Sciences
Division, SRI International,Menlo Park, California 94025 [M. l. D.,
P. D. H.]
ABSTRACT
Retinole acid (RA) and its synthetic analogues, retinoids, have
shownpromising results in the prevention of epithelial
carcinogenesis and in thetreatment of acute promyelocytic leukemia
and various proliferatile skindisorders. Retinoid action on gene
regulation is mediated by three distinctnuclear retinole acid
receptor subtypes, RA receptors a, 0, and ->. The
existence of multiple RA receptors has raised the possibility
that receptorsubtype-specific retinoids with reduced side effects
can be developed. To
analyze the activity of retinoids at the molecular level, we
used a receptoractivation assay. RA and 22 retinoids were compared
on the three receptorsubtypes. We found the a receptor to be least
sensitive to activation byRA and the y receptor to be most
sensitive. Compared with RA, one ofthe retinoids showed increased
activity for the a and ,? receptors. Threeretinoids revealed no
gene activation activity and showed no antagonisticeffects when
assayed in the presence of RA. Surprisingly, several of
theretinoids were efficient activators of the ßand 7 receptors but
pooractivators or nonactivators of the a receptor. Our data
demonstrate thatthe three RA receptor subtypes have differential
ligand activation specificities and that the design of receptor
subtype-selective retinoids is
possible.
INTRODUCTION
RA3 and its synthetic analogues (retinoids) affect processes
as diverse as growth, differentiation, morphogenesis,
reproduction, and vision (for a review, see Refs. 1-3) and suppress
andreverse malignant transformation induced by either
chemicalcarcinogens or ionizing radiation (4, 5). The beneficial
effectsof retinoids in the treatment of cancer patients is best
reflectedby a recent clinical study by Hong et al. (6). The
syntheticretinoid 13-c/'s-retinoic acid (isotretinoin),
administered in high
doses, significantly reduced the occurrence of second
primarytumors in patients with squamous cell carcinomas of the
headand neck. The full potential of retinoids as
chemopreventativeagents has been hampered by their undesirable
systemic sideeffects (7) and teratogenicity (8). Although many
syntheticretinoids have been evaluated (9), analysis of the
structure-activity relationships has been difficult because of the
differences in activities of individual retinoids observed in the
variousin vitro and in vivo test systems (9, 10). These differences
cannotbe explained solely by pharmacokinetic arguments and
probablyresult from different modes of retinoid action in the
varioussystems.
The recent discovery of nuclear RARs (11-16) as mediatorsof RA
action on gene regulation provides us with the opportunity to
investigate the effects of retinoids at the molecular level.The
RARs belong to a large family of ligand-activated transcrip-
Received 4/18/91; accepted 7/9/91.The costs of publication of
this article were defrayed in part by the payment
of page charges. This article must therefore be hereby marked
advertisement inaccordance with 18 U.S.C. Section 1734 solely to
indicate this fact.
1This work was supported in parts by NIH Grants DK35083 and
CA50676
(M. P.) and 32428 (M. I. D.). J. M. L. and M. H. were supported
by a fellowshipfrom the Deutsche Forschungsgemeinschaft.
1To whom requests for reprints should be addressed, at La Jolla
Cancer
Research Foundation, 10901 North Torrey Pines Road. La Jolla. CA
92037.'The abbreviations used are: RA. rctinoic acid; CAT,
chloramphenicol acetyl
transferase; ER, estrogen receptor; ERE, estrogen responsive
element: RAR.retinoic acid receptor; rfRARE, retinoic
acid-responsive element of the RAR/3promoter; tk, thymidine
kinase.
tion factors that interact specifically with cognate DNA
elements (i.e., hormone response elements; for a review see Refs.17
and 18). Three retinoic acid receptors, RAR«, ß,and 7,encoded by
distinct genes, have been identified in humans (lilo). In addition,
a related group of receptors (RXRa, ß,7) wasisolated, which are
activated by retinoids as well (19). Fromeach of the three RAR
genes a number of receptor isoformscan be generated that contain
different amino terminal sequences but are identical in their DNA-
and ligand-bindingdomains (15, 16, 20-23). The identification of
this complexvariation in RARs suggests that many of the diverse
biologicaleffects of retinoids may be due to their differential
receptoraffinities, combined with different temporal and spatial
expression patterns of receptor subtypes and isoforms (24-26).
To elucidate the molecular mechanisms underlying
retinoidtranscriptional activity and to develop more specific
retinoidshaving fewer undesirable side effects, we investigated the
possibility of identifying receptor-selective retinoids as well
asantagonists. To achieve this goal, an assay system was developed
in which retinoids were evaluated for their
transcriptionalactivation capabilities. Because apparently all
mammalian celllines contain endogenous RARs (Ref. 27),4 hybrid
receptorswere constructed that consist of the ligand-binding domain
andthe carboxy-terminal portion of either RARa, ß,or 7, and
theDNA-binding domain and amino-terminal portion of the ER.These
hybrid receptors showed retinoid specificities identicalto those of
the wild-type RARs (28). RA and 22 conformation-ally restricted
retinoids were evaluated for their transcriptionalactivation
activity. The identification of several retinoids withreceptor
subtype-selective activity patterns shows for the firsttime that
RARs can be activated differentially and that thedevelopment of
receptor-specific retinoids is possible.
MATERIALS AND METHODS
Chemicals. All-/rani-retinoic acid (Rl of Fig. 3) was purchased
fromSigma Chemical Co. (St. Louis, MO).
(£)-4-[2-(5,6,7,8-Tetrahydro-5,5,8,8-tetramethyl-2-naphthalenyl)propenyl]benzoic
acid (R2) wasprepared by the method of Loeliger et al. (29). The
following retinoids(R3-R10), which are analogues of R2, were
prepared by similar methods, which are described elsewhere (30,
31):
(£>4-[2-(5,6,7,8-tetrahy-dro-5,5-dimethyl-2-naphthalenyl)propenyljbenzoic
acid (R3) (30);
(£)-7-[1-(4-carboxyphenyl)propen-2-yl]-3,4-dihydro-2//-1
-benzothiopyran(R4) (30);
(E)-4-[2-(5,6,7,8-tetrahydro-8,8-dimethyl-2-naphtha-lenyl)propenyl]benzoic
acid (R5) (30);
(£>6-[l-(4-carboxy-phenyl)propen-2-yl]-4,4-dimethyl-3,4-dihydro-2//-1
-benzopyran (R6)(31);
(E)-6-[l-(4-carboxyphenyl)-4,4-dimethylpropen-2-yl]-3,4-dihydro-2//-1
-benzothiopyran (R7) (31);
(£)-4-[2-(3-f-butyi-4-methoxy-phenyl)propenyl]benzoic acid (R8)
(30); (£)-4-|2-[4-(3-methylbu-tyl)thiophenyl]propenyl|benzoic acid
(R9) (30); and
(£)-4-[2-(4,5,6,7-tetrahydro-4,4-dimethylbenzo[6]thienyl)propenyl]
benzoic acid (RIO)(30).
(£)-4-[l-(5,6,7,8-Tetrahydro-5,5,8,8-tetramethyl-2-naphtha-lenyl)propen-2-yl]benzoic
acid (RII) was prepared as described
(32).(£)-6-[2-(4-Carboxyphenyl)propenyl]-4,4-dimethyl-3,4-dihydro-2//-l-benzothiopyran
(R12) was prepared by a similar method (30). Thesyntheses of
retinoids R13 and R14 have not been previously reported
4 Unpublished observations.
4804
on June 30, 2021. © 1991 American Association for Cancer
Research. cancerres.aacrjournals.org Downloaded from
http://cancerres.aacrjournals.org/
-
RETINOIDS WITH RECEPTOR-SELECTIVE ACTIVITIES
and so will be described here.
(E)-4-[l-(5,6,7,8-Tetrahydro-5,5,8,8-tetramethyl-4-methoxy-2-naphthalenyl)propen-2-yl]benzoic
acid (R 13)(30) was prepared by the following route: (a)
introduction of functionality at the 4-position of the
tetrahydronaphthalene ring by nitration(HNO3, H2SO4, MeNO2,
0°C;79%) of ethyl 5,6,7,8-tetrahydro-5,5,8,8-
tetramethyl-2-naphthalenecarboxylate (33) at the 3-position,
followedby reduction [H2, Pd(C), Et2O] and acetylation (Ac2O,
pyridine, Et2O;100%) to give the amid, and nitration (NO2BF4, MeCN,
0-20°C;74%)
at the 4-position, cleavage (KOH, aq. EtOH; aq. HC1; 97%) of
theacetyl group, removal of the 3-amino group by deamination
(n-C6H,3ONO, CF3CO2H/EtOH; H3PO2; 81 %), and reduction [H2,
Pd(C),EtOH; 100%] of the 4-nitro group to the amine to give
4-amino-5,6,7,8-tetrahydro-5,5,8,8-tetramethyl-2-naphthalenecarboxylic
acid; (b) conversion of the 2-carboxylic acid function to the
phosphonium salt byreduction (LiAlH4, tetrahydrofuran, 20°C;aq.
NaOH), acetylation
(Ac2O, pyridine, Et2O), hydrolysis of the benzyl acetate
(K2CO3,MeOH, 20°C;76% overall) to the alcohol, and treatment with
(C6H5)3P(xylene, 80°C;89%); (c) formation of the propen-2-yl bond
system byWittig reaction (-10°C to 20°C;58%) of the ylid derived
from treatment of the phosphonium salt with n-BuLi
(tetrahydrofuran, -35°Cto—10°C)and ethyl 4-acetylbenzoate, and
Chromatographie separation
(35% EtOAc/hexane, silica gel; 41%) of the £isomer; (d)
hydrolysis(KOH, aq. diethyleneglycol, 40°C;aq. HC1; 83%) of the
acid and amine
protecting groups; and (e) introduction of the 4-methoxy group
bynonaqueous diazotization (n-C6H13ONO, CF3CO2H/EtOH) andaqueous
thermal decomposition (aq. NaOH; aq. H2SO4) of the
resultantdiazonium salt to give the phenol, which was purified as
the ethyl ester(CH3CHN2, Et2O; 56% overall), followed by
methylation of the phenolunder phase-transfer conditions (Me2SO4,
(n-Bu)4NI, 50% aq. NaOH,CH2C12;94%) (34), and ester hydrolysis
(NaOH, aq. MeOCH2CH2OH,20°C;aq. HC1; 100%). This rather complex
route was employed because
of the availability of the starting materials and was not
process developed.
(£)-4-[3-(5,6,7,8-Tetrahydro-5,5,8,8-tetramethyl-2-naphthal-enyl)-2-buten-2-yl]benzoic
acid (R14),
Ã-ranÃ--4-[2-(5,6,7,8-tetrahydro-5,5,8,8-tetramethyl-2-naphthalenyl)cyclopropyl]benzoic
acid (R 15),and
4-(5,6,7,8-tetrahydro-5,5,8,8-tetramethyl-2-anthracenyl)benzoicacid
(R16) were synthesized as described (32), as were
(4-(4-azido-5,6,7,8-tetrahydro-5,5,8,8-tetramethyl-2-anthracenyl)benzoic
acid(R 17) and
(£>3-azido-4-[2-(5,6,7,8-tetrahydro-5,5,8,8-tetramethyl-2-naphthalenyl)propenyl]benzoic
acid (R19) (35).
4-(5,6,7,8-Tetrahydro-5,5,8,8-tetramethyI)naphtho[2,3-b]thiophen-2-yl)benzoic
acid (R18) was prepared from the methyl ester of thecorresponding
3-bromo analogue, which was used as an intermediatein another
synthesis. This intermediate was obtained by (a) introductionof
ring functionality by Friedel-Crafts alkylation of
benzo[¿>]thiophenewith toluene (A1C13,CH2C12,80*C) (36) at the
2-position to give the 4-
methylphenyl and 2-methylphenyl isomers, followed by
Chromatographie isolation of the 4-methylphenyl compound (20%) and
bromi-nation (CHC13; 92%) at the 3-position of its
benzo[6]thiophene ring,and cyclialkylation (cat. A1C13,CH2C12,
-10°C; 44%) of the resultant
2-aryl-3-bromobenzo[2>]thiophene with
2,5-dichloro-2,5-dimethylhex-ane; and (b) conversion of the methyl
group to the methyl ester byradical bromination
[A'-bromosuccinimide, (C6H5CO2)2,CC14,hu), dis
placement of the benzylic bromide by acetate (CH3CO2K,
HCONMe2;70%), and transesterification to give the benzyl alcohol
(K2CO3, EtOH,80°C;45% overall), which was oxidized using Corey's
procedure(Mn02, NaCN, HOAc, MeOH, 20°C;90%) (37) to give the
methylester. The 3-bromo group was removed by hydrogenolysis [H2,
Pd(C),Et3N, EtOAc; 95%), and the methyl ester removed (KOH, aq.
EtOH;aq. H2SO4; 100%) to give R18.
6-(5,6,7,8-Tetrahydro-5,5,8,8-tetramethyl-2-naphthalenyl)-2-naphthalenecarbo
xylic acid (R20),
6-(5,6,7,8-tetrahydro-3,5,5,8,8-pen-tamethyl-2-naphthalenyl)-2-naphthalenecarboxylic
acid (R22), and
6-(5,6,7,8-tetrahydro-5,5,8,8-tetramethyl-2-naphthalenyl)-5-methyl-2-naphthalenecarboxylic
acid (R23) were synthesized as described (32),using methodology
that was also employed to synthesize
6-(5,6,7,8-tetrahydro-8,8-dimethyl-2-naphthalenyl)-2-naphthalenecarboxylic
acid(R21) (30). All retinoids were fully characterized ('H nuclear
magnetic
resonance spectrometry, infrared and UV spectroscopy, melting
point,and elemental analysis) and were of at least 99% purity by
high-
performance liquid chromatography. Stock solutions (10 2 M) in
dimethyl sulfoxide were maintained at -20°C. Dilutions were made
in
cell culture medium prior to usage.Transient Transfection. For
all transient transfection assays we used
green monkey kidney cells (CV-1 ) grown in Dulbecco's modified
Eagle's
medium supplemented with 10% fetal calf serum. The fetal calf
serumhad been pretreated with charcoal (12 mg/ml serum) to remove
retinoids. Transient transfections using the calcium phosphate
precipitation procedure were performed as described (Ref. 28 and
referencestherein) with the following modifications. Cells (7 x
IO4)were seededin a volume of 1 ml/well in 24-well tissue culture
plates (Costar,Cambridge, MA). In each well, transfection mixtures
contained a totalof 1 /jg supercoiled plasmid DNA including 50 ng
of receptor expressionvector and 100 ng of reporter plasmid for
each well to be transfected.Cotransfection of 300 ng
/3-galactosidase expression vector pCHllO(Pharmacia, Piscataway,
NJ) served to correct for variation in transfection and harvesting
efficiency. After overnight incubation, mediumexchange, and
retinoid treatment for 24 h, lysates were obtained bythree in situ
freeze-thaw cycles. Cell debris was removed by centrifu-gation.
Quantitation of CAT activity was performed with a
sensitiveextraction-liquid scintillation procedure described
elsewhere (Ref. 28and references therein). Linearity of the assays
with time and ratio ofCAT: /3-galactosidase activity was
established.
Hybrid Receptor Constructs. RAR/3 was previously referred to
asRAR«(14). Isolation of RAR7 (21), as well as the isolation of
theRAR«ligand-binding domain and the construction of ER-RAR
hybridreceptors using the polymerase chain reaction, have been
described(28). Briefly, standard polymerase chain reactions (10
cycles, denatu-ration at 94'C for 1.5 min, annealing for 2 min at
37'C, elongation for2 min at 72"C) were carried out using 1 Mg of
the corresponding
receptor complementary DNA cloned in pBluescript (Stratagene,
LaJolla, CA). The oligonucleotide primers were synthesized
according tothe complementary DNA sequence but contained an
artificially introduced endonuclease restriction site (i.e., BamHl
site). As a commonlink-up site of the NH2-terminal estrogen
receptor half and the COOH-terminal half of the RAR subtypes, we
have chosen a site in the hingeregion, 71 bases upstream of the
beginning of the ligand-bindingdomain.
RESULTS
RAR Hybrid Receptors Show Subtype Differential Response.Because
mammalian cell lines generally contain variousamounts of endogenous
RARs (27, 38), it is often impossibleto distinguish activation of
the transgene by the endogenousreceptor(s) from activation by the
cotransfected target RAR.Hybrid receptors that contain the
ligand-binding domains ofRARs and the DNA-binding domain of the
less ubiquitouslyexpressed receptor allow the usage of a hybrid
receptor-specificreporter gene for measuring retinoid activities
that cannot beactivated by endogenous RARs. An example is shown in
Fig.
ßRARE tk-CAT ERE tk-CAT
RA RA
Fig. 1. Reporter gene response to endogenous RAR activity of
CV-1 cells. CV-1 cells were transiently transfected with either the
ßRARE-tk-CAT(38) or theERE-tk-CAT reporter gene (41), treated for
24 h with RA as indicated, andassayed for CAT activity as described
in "Materials and Methods."
4805
on June 30, 2021. © 1991 American Association for Cancer
Research. cancerres.aacrjournals.org Downloaded from
http://cancerres.aacrjournals.org/
-
RETINOIDS WITH RECEPTOR-SELECTIVE ACTIVITIES
1, CV-1 cells reveal endogenous RAR activity when
transfectedwith a reporter construct containing the ßRARE(38) but
notwhen a reporter gene is used that contains an ERE. Therefore,we
employed hybrid receptors that contained the DNA-bindingdomain and
amino-terminal half of the human ER linked tothe ligand-binding
domain and carboxy-terminal portion of thethree different RAR
subtypes a, ß,or 7, in order to be able torigorously determine
receptor-specific retinoid activities. Domain swapping among
nuclear receptors is possible because theligand-binding domains of
nuclear receptors maintain theirspecificity when linked to the
DNA-binding domains of otherreceptors (14,39) and even when linked
to members of differenttranscription factor families (40). In
addition we previouslydemonstrated that hybrid receptors containing
the RAR ligand-binding domain were activated specifically by RA and
otherretinoids (28, 39). The linkage site for all the
hybrid-receptorconstructs was at an introduced restriction site in
the hingeregion located between the DNA-binding and
ligand-bindingdomains as described in "Materials and Methods." As
the
reporter gene, a CAT construct (ERE-tk-CAT) was used
thatcontained an ERE at the —105position of the herpes
simplex
virus tk promoter (41).Transcriptional activation of the three
hybrid receptors by
RA at concentrations ranging from 0.1 nM to 1 I¿Mshowed
adifferential response (Fig. 2A). The y and ßreceptors were
themost sensitive and the a receptor the least sensitive. In
addition,all three receptors showed different basal level
activities in theabsence of ligand, with y having the highest and a
the lowestlevel of constitutive activity. Both results are
consistent withdata obtained for the wild-type receptors (16, 21).
To comparethe retinoid activities in the transcriptional activation
assay, thedata were normalized by subtraction of the basal level
activityand by expressing the amount of transcriptional activation
as apercentage of the activation obtained with each receptor at
10~6
M RA (Fig. 2B).The fact that the three hybrid receptors yielded
distinct
induction profiles indicated that the structural differences
observed in their ligand-binding domains confer differential
responsiveness to RA. These data suggested to us the possibilityof
finding retinoids with enhanced selective responsiveness toone of
the receptors.
Synthetic Retinoids Reveal Receptor-Selective Activation
Profiles. We compared the receptor-mediated transcriptional
activation activity of 22 retinoids with that of RA. These
retinoidswere conformationally restricted analogues of RA in
whichselected bonds of the polyolefinic chain of the retinoid
skeletonwere incorporated into aromatic ring systems. Because
thesecompounds have fewer degrees of freedom, they provide
moreuseful information about the geometry of the ligand-bindingsite
than the more conformationally flexible RA. The structuresof these
analogues, which are shown in Fig. 3, fall into twogeneral classes,
the 4-substituted benzoic acids R2-R19 havingthe 11,13-double bonds
of RA replaced by a phenyl ring andthe 6-substituted
2-naphthalenecarboxylic acids R20-R23 having the 9,11,13-double
bond system of RA replaced by a naphthalene ring. All analogues
have an aromatic ring (phenyl orthienyl) replacing the 5,7-double
bond system. Modificationsin the /3-cyclogeranylidene ring included
removal of the gem-dimethyl groups at the 1- and 4-positions of the
ring, replacement of carbons at these positions with more polar
atoms(oxygen and sulfur), and species having the 2-3 and 1-6
bondsbroken. Other modifications were made in the region of the
9-double bond, where the methyl group was shifted to the
adjacent10-posi t ion. an additional methyl group was placed at the
10-position, the bond system was replaced by a cyclopropyl
ring,
10 ' 10 ° 10
RA cone. (M)
Fig. 2. Retinole acid-dependent transcriptional activation of
ER-RAR hybridreceptors. CV-1 cells were transiently transfected
with expression plasmids forthe hybrid receptors ER-RAR«,ß,or y,
the reporter gene ERE-tk-CAT, and (forinternal standardization)
with the tÃ--galactosidaseexpression plasmid p( III 10.Cells were
treated subsequently for 24 h with increasing concentrations of RA,
asindicated, and were assayed for CAT activity. A, dose-response
curve for receptor-mediated transactivation by RA. Points, average
of five experiments; B, receptoractivation expressed as a
percentage of maximal induction (measured at 10 " \i
RA) after constitutive receptor activity was substracted.
or the incorporation of the bond system into a phenyl or
thienylring. The effect of increased steric bulk on biological
activitywas assessed using methoxy, azido, and methyl group
substit-uents at various aromatic ring positions.
The transactivation assay results are summarized in Fig. 3.The
data represent the average of three independent experiments, and
the SD at retinoid concentrations that resulted insubstantial
(>25%) receptor activation typically did not exceed10%. To
ensure that the hybrid receptors faithfully representedRAR subtype
specificity we also evaluated all retinoids for theiractivity with
RAR/3 and observed no substantial differencesfrom ER-RAR0 (data not
shown). One of the retinoids, R16,showed clearly increased activity
for the 0-receptor and a slightbut significant increase for the
a-receptor compared with thatof RA (Fig. 4A). The high activity was
consistent with highactivities reported for R16 in other in vitro
systems (9). Threeretinoids (R9, R14, and R23) displayed no gene
activationactivity, which again was consistent with their lack of
activityin in vitro assay systems (9). When these compounds
wereassayed in the presence of RA, they did not display any
antagonist activity either, suggesting that their affinity for the
receptors is low (data not shown).
Several retinoids displayed differential activity for the
threereceptors. Generally, the greater differences in
transcriptionalactivation activity were observed between ßand a
and betweeny and a. The strongest examples are R15, R19, R20, and
R22,which were efficient activators of the ßand y receptors
atcritical concentrations but poor activators or nonactivators
ofthe a receptor (Fig. 4B). In contrast, activation of ßby 10~7and
10~6 M R21 was 2-fold higher than that for the a or 7
receptor (Fig. 4C). This was the only example in the two
seriestested in which the activation pattern of 7 was clearly
closer tothat of a than to .1 In general, our studies indicated
that thetranscriptional activation assay described here will permit
theidentification of retinoids with differential receptor
activationactivity. Receptor activation was dependent on retinoid
concentration, except in two cases (R7 and R13), in which
activationof the ßreceptor was higher at IO"7M than at IO"6M.
4806
on June 30, 2021. © 1991 American Association for Cancer
Research. cancerres.aacrjournals.org Downloaded from
http://cancerres.aacrjournals.org/
-
RETINOIDS WITH RECEPTOR-SELECTIVE ACTIVITIES
Fig. 3. Summary of transcriptional activation characteristics of
RA and 22 retinoids.Structures of RA (Rl) and the retinoids R2
toR23 are shown. The receptor activation capacities were evaluated
as described in Fig. 2.Retinoid-induced receptor activation is
expressed as a percentage of maximal inductionby RA after
constitutive receptor activity wassubstracted. The activation
values were dividedinto five activity groups, as indicated by
thesizes of the columns: 81%. The data represent theaverage of
three experiments. One hundred %receptor activation represents the
activity observed for each receptor in the presence of 10~6
M RA.
DC retinoid cone (M)RETINOID 2 10'°109 10 8 10 7 10 10'° 109
10e 107 106
roroptcr activation ('
• •81
DISCUSSION
We have analyzed here retinoids as inducers of retinoic
acidreceptor subtypes, as measured by gene activation. To
facilitatethe interpretation of the retinoid induction data, we
have employed hybrid receptors in the transcriptional activation
assaythat contain the NH2-terminal half of the ER and COOH-
terminal half of the different RAR subtypes. When comparedto
wild-type RARs we observe no significant difference in theresponse
of these hybrid receptors to RA and the 22 retinoidstested. Thus,
at the molecular level, this system is likely tomeasure the major
biological activity of individual RARs. Surprisingly, by having
tested only a limited number of compounds,we were able to obtain
clear evidence that individual retinoidscan have striking
differences in their activation capacity for thedifferent receptor
subtypes. This may at least partially explainthe different activity
profiles in various biological assays observed previously for the
same retinoids (10). The variations indifferent assay systems may
thus reflect the activities of distinctRARs or various combinations
of them. In our transcriptionalactivation assay we used green
monkey kidney CV-1 cells. Many
other cell lines can certainly be used. It will be of interest
to seewhether cellular factors, including the retinoid binding
proteinsCRBP and CRABP and retinoid-modifying enzymes present
insome cell lines, can influence the transcriptional
activationcapacity of certain retinoids. From enzymatic activities
positiveas well as negative effects can be expected. For CRABP,
recentevidence indicates that it may reduce the effective
intracellularconcentration of RA (42); thus similar effects can be
expectedfor other CRABP-binding retinoids.
Although the three-dimensional structure of the RAR
ligand-binding pockets has not yet been elucidated, analysis of
thesequence homology of the RARs indicates that the ligand-
binding domains of RARi and ßare more closely related toeach
other than to RARa (11-16). This finding is consistentwith our
observation that some of these retinoids showed activation profiles
that were more similar and higher for RAR/3and 7 than for RARa.
Interestingly, the inverse situation inwhich a compound showed
higher activity with RARa thanwith ßand y was not found. These
results suggest that at leastfor the compounds analyzed here, the
ßand 7 receptor ligand-
4807
on June 30, 2021. © 1991 American Association for Cancer
Research. cancerres.aacrjournals.org Downloaded from
http://cancerres.aacrjournals.org/
-
RETINOIDS WITH RECEPTOR-SELECTIVE ACTIVITIES
B
Fig. 4. Dose-response curves for retinoids. The receptor
activation patterns were evaluated with the ER-RAR hybrid receptors
(«,0, y) as described for Rl in Fig.2. One hundred % receptor
activation represents the activity measured in the presence of
IO"6M RA. A, retinoid R16; A, receptor subtype specific retinoid
R15, R19,
R20, and R22; C, retinoids R21.
binding domains are more tolerant of the tetraene chain-modified
retinoids than is the a receptor ligand-binding domain.The
ligand-binding domain of the RARs has been conservedalmost
completely during mammalian evolution, showing a 99to 100% identity
between human and mouse (20). Thus, thedifferences in retinoid
activities appear to correlate with theminor structural differences
in this domain among the receptorsubtypes. Northern blot and in
situ hybridization analysis demonstrated ubiquitous expression of
RARa, whereas RAR/3 andy expression is spatially and temporally
regulated (26-28). Theavailability of RAR/3/7-specific retinoids
allows the biologicalfunctions of the ubiquitously expressed RAR«
to be distinguished from those of RAR/3 and y.
Our study indicates that receptor-selective retinoids can
beidentified. RARa appears to be far more sensitive to variationsin
the structural geometry of the retinoids in the
4-substitutedbenzoic acid and 6-substituted 2-naphthalenecarboxylic
acidclasses than RAR/? and RAR7 are. Modifications at the
4-position of the retinoid skeleton appear to contribute to
thisselectivity. An independent study, analyzing modifications
of6'-substituted naphthalene-2-carboxylic acid analogues in
moredetail, has also revealed striking selectivity of this class of
c/'.v-
RA analogues for RAR/3 and RAR7 over RARa.5 Our results
are also consistent with other reports of receptor
selectivity.For example, benzoic acid R2 is reported to have 50% of
thebinding affinity for recombinant RARa and 70% of the affinityfor
RAR/3 that RA has (43). R2 was also found to induce RAR/3mRNA in
S91-C2 melanoma cells less efficient than RA (44).This is
consistent with our results that show R2 as a lessefficient
activator than RA of these two receptors. 2-Naphtha-lenecarboxylic
acid R20, which we found is less effective thanRA at RARa and RAR/3
activation, is also reported to bind toboth recombinant receptors
less efficiently (43). In addition,our most efficient
transcriptional activator, R16, had the highest binding affinity
among 13 retinoids evaluated (43). RA andR2 were also among a
series of eight retinoids evaluated forstimulation of RAR-dependent
transcription using a retinoicacid-responsive reporter gene
(TRE3-tk-CAT) in cotransfected
*G. Graupner, G. Malle, G. Lang, M. Pruniéras,and M. Pfahl.
6'-Substitutednaphthalene-2-carboxylic acid analogs, a new class of
retinoic acid receptorsubtype-specific ligands. Biochem. Biophys.
Res. Commun., in press, 1991.
CV-1 cells (45). Although clear differences in activation
activities of compounds for RAR subtypes were observed, the
retinoids analyzed were active with all three receptor
subtypes.
In conclusion, our molecular approach has yielded resultsthat
allow us to further select and develop receptor-specificretinoids.
Such compounds will be valuable tools for the dissection of the
complex biological pathways of RA action. Theefficacy of retinoids
as anticancer drugs is best reflected by tworecent studies, one
showing the prevention of second primarycarcinomas in patients with
squamous cell carcinomas of thehead and neck (6), the other showing
complete remissions inpatients with acute promyelocytic leukemia
upon treatmentwith retinoic acid (46). However, intolerable side
effects due torelatively high doses and length of the treatment led
to highdropout rates. This underscores the importance of
developingbetter tolerated retinoids which might be accomplished by
theusage of receptor subtype-specific retinoids. Together with
theknowledge of the receptor distribution in normal and
diseasedtissue, receptor-specific retinoids may in the future
permit thedesign of efficient retinoid therapies with minimal side
effects.The test systems described here can be easily extended to
otherretinoid receptors such as the RXRs, which appear to be
activated by RA derivatives rather than by RA itself ( 19).
ACKNOWLEDGMENTS
We thank Elmira Khodapanah for her excellent technical
assistance.
REFERENCES
1. Roberts, A. B., and Sporn, M. B. Cellular biology and
biochemistry of theretinoids. In: M. B. Sporn, A. B. Roberts, and
D. S. Goodman (eds.), TheRetinoids, Vol. 2, pp. 210-286. Orlando,
FL: Academic Press, Inc., 1984.
2. Lotan, R. Effect of vitamin A and its analogs (retinoids) on
normal andneoplastic cells. Biochim. Biophys. Acta, 605: 33-91,
1980.
3. Thaller,
-
RETINOIDS WITH RECEPTOR-SELECTIVE ACTIVITIES
R. M., Schantz, S. P., Kramer, A. M., Lotan, R., Peters, L. J.,
Dimer>', I.W., Brown, B. W., and Goepfert, H. Prevention of
second primary tumorswith isotretinoin in squamous-cell carcinoma
of the head and neck. N. Engl.J. Med., 323: 795-801, 1990.
7. Moon, R. G., and Itri, L. M. Retinoids and cancer. In: M. B.
Sporn, A. B.Roberts, and D. S. Goodman (eds.). The Retinoids, Vol.
2, pp. 327-371.New York: Academic Press, 1984.
8. Lammer, E. J., Chen, D. T., Hoar, R. M., Agnisti, N. D.,
Benke, P. J.,Braun, J. T., Curry, C. J., Fernhoff, P. M., Grix,
Jr., A. W., Lott, I. T.,Richard, J. M., and Shyan, C. S. Retinoic
acid embryopathy. N. Engl. J.Med., 313: 837-841, 1985.
9. Dawson, M. I., Chao, W., Hobbs, P. D., and Delair, T. The
inhibitory effectsof retinoids on the induction of ornithine
decarboxylase and the promotionof tumors in mouse epidermis. In: M.
I. Dawson and W. H. Okamura (eds.),Chemistry and Biology of
Synthetic Retinoids, pp. 385-466. Boca Raton,FL: CRC Press, Inc.,
1990.
10. Sporn, M. B., and Roberts, A. B. Biological methods for
analysis and assayof retinoids—relationship between structure and
activity. In: M. B. Sporn,A. B. Roberts, and D. S. Goodman (eds.),
The Retinoids, Vol. 2, pp. 235-279. Orlando, FL: Academic Press,
Inc., 1984.
11. Petkovich, M., Brand, N. J., Krust, A., and Chambón, P. A
human retinoicacid receptor belongs to the family of nuclear
receptors. Nature (Lond.), 330:444-450, 1987.
12. ( i ignore, V., Ong, E. S., Segui, P., and Evans, R. M.
Identification of areceptor for the morphogen retinoic acid. Nature
(Lond.), 330: 624-629,1987.
13. Brand, N., Petkovich, M., Krust, A., de The, H., Marchio,
A., Tiollais, P.,and Dejean, A. Identification of a second human
retinoic acid receptor.Nature (Lond.), 332:850-853. 1988.
14. Benbrook, D., Lernhardt, E., and Pfahl, M. A new retinoic
acid receptoridentified from a hepatocellular carcinoma. Nature
(Lond.), 333: 669-672,1988.
15. Krust, A., Kastner, P. H., Petkovich, M., Zelent, A., and
Chambón, P. Athird retinoic acid receptor, hRAR-i. Proc. Nati.
Acad. Sci. USA, 86: 5310-5314, 1989.
16. Giguere, V., Shago, M., Zirngibl, R., Tate, P., Rossant, J..
and Varmuza, S.Identification of a novel isoform of the retinoic
acid receptor-y expressed inthe mouse embryo. Mol. Cell. Biol., 10:
2335-2340, 1990.
17. Evans, R. M. The steroid and thyroid hormone superfamily.
Science (Washington DC), 240: 889-895, 1988.
18. Green, S., and Chambón, P. Nuclear receptors enhance our
understandingof transcription regulation. Trends Genet., 4:
309-314, 1988.
19. Mangelsdorf, D. J., Ong, E. S., Dyck, J. A., and Evans, R.
M. Nuclearreceptor that identified a novel retinoic acid response
pathway. Nature(Lond.), 345: 224-229, 1990.
20. Kastner, P. H., Krust, A., Mendelsohn, C., Gamier, J. M.,
Zelent, A., Leroy,P., Staub, A., and Chambón, P. Murine isoforms
of retinoic acid receptor ywith specific patterns of expression.
Proc. Nati. Acad. Sci. USA, 87: 2700-2704, 1990.
21. 1dimani], J. M., Hoffmann, B., and Pfahl, M. Genomic
organization of theretinoic acid receptor y gene. Nucleic Acids
Res., 19: 573-578, 1991.
22. Leroy, P., Krust, A., Zelent, A., Mendelsohn, C., Gamier,
J-M., Kastner, P.,Dierich, A., and Chambón, P. Multiple isoforms
of the mouse retinoic acidreceptor a are generated by alternative
splicing and differential induction byretinoic acid. EMBO J., 10:
59-69, 1991.
23. Zelent, A., Mendelsohn, C., Kastner, P., Krust, A., Gamier,
J-M., Ruffenach,F., Leroy, P., and Chambón, P. Differentially
expressed isoforms of themouse retinoic acid receptor .t are
generated by usage of two promoters andalternative splicing. EMBO
J., 10: 71-81, 1991.
24. Dolle, P., Ruberie, E., Kastner, P., Petkovich, M., Stoner,
C. M., Gudas, L.J., and Chambón, P. Differential expression of
genes encoding a, ßand yretinoic acid receptors and CRABP in the
developing limbs of the mouse.Nature (Lond.), 342: 702-704,
1989.
25. Ruberie, E., Dolle, P., Krust, A., Zelent, A., Morriss-Kay,
G., and Chambón,P. Specific spatial and temporal distribution of
retinoic acid receptor ytranscripts during mouse embryogenesis.
Development (Camb.), ¡08:213-222, 1990.
26. Dolle, P., Ruberie, E., Leroy, P., Morriss-Kay, G., and
Chambón, P. Retinoic
acid receptors and cellular retinoid binding proteins.
Development (Camb.),7/0:1133-1151. 1990.
27. Sucov, H. M., Murakami. K. K., and Evans. R. M.
Characterization of anautoregulated response element in the mouse
retinoic acid receptor type .',gene. Proc. Nati. Acad. Sci. USA,
87: 5392-5396, 1990.
28. Pfahl, M., Tzukerman, M., Zhang, X-K., Lehmann, J. M.,
Hermann, T.,Wills, K. N., and Graupner, G. Rapid procedures for
nuclear retinoic acidreceptor cloning and their analysis. Methods
Enzymol., 153: 256-270, 1990.
29. Loeliger, P., Bollag, W., and Mayer, H. Arotinoids, a new
class of highly-active retinoids. Eur. J. Med. Chem., IS: 9-15,
1980.
30. Schiff, L. J., Okamura, W. H., Dawson, M. I., and Hobbs, P.
D. Structurebiological activity relationships of new synthetic
retinoids on epithelial differentiation of cultured hamster
trachea. In: M. I. Dawson and W. H.Okamura (eds.). Chemistry and
Biology of Synthetic Retinoids, pp. 308-363. Boca Raton, FL: CRC
Press, Inc., 1990.
31. Dawson, M. I., Hobbs, P. D., Derdzinski, K., Chan, R. L.,
Gruber, J., Chao,W., Smith, S., Thies, R. W., and Schiff, L. J.
Conformationally restrictedretinoids. J. Med. Chem., 27: 1516-1531,
1984.
32. Dawson, M. I., Hobbs. P. D., Derdzinski, K. A., Chao. W..
Frenking, G.,Loew, G. H., Jetten, A. M., Napoli, J. L., Williams,
J. B., Sani, B. P., Wille,J. J., Jr., and Schiff, L. J. Effect of
structural modifications in the C7-C11region of the retinoid
skeleton on biological activity in a series of aromaticretinoids.
J. Med. Chem., 32: 1504-1517, 1989.
33. Myhre, P. C., and Schubert, W. M. Isolation and proof of
structure
ofl,l,4,4-tetramethyl-6-/-butyl-l,2,3,4-tetrahydronaphthalene. J.
Org. Chem.,25:708-711,1960.
34. McKillop, A., Fiaud, J. C., and Hug, R. P. The use of
phase-transfer catalysisfor the synthesis of phenol ethers.
Tetrahedron, 30: 1379-1382, 1974.
35. Sani, B. P., Wille, J. J., Jr., Dawson, M. I., Hobbs, P. D.,
Bupp, J., Rhee,S., Chao, W., Dorsky, A., and Morimoto, H.
Biologically active aromaticretinoids bearing azido
photoaffinity-labeling groups and their binding tocellular retinoic
acid-binding protein. Chem.-Biol. Interact.. 75: 293-304,1990.
36. Clark, P. D., Clarke, K., Ewing, D. F., and Scrowston, R. M.
Additionreactions of benzo[ojthiophen. Part 1. Self-addition of
simple aromatichydrocarbons. J. Chem. Soc. Perkin Trans. I, /:
677-685, 1980.
37. Corey, E. J., Gilman, N. W., and Ganem, B. E. New methods
for theoxidation of aldehydes to carboxylic acids and esters. J.
Am. Chem. Soc., 90:5616-5617, 1968.
38. Hoffmann, B., Lehmann, J. M., Zhang, X-k., Hermann, T.,
Husmann, M.,Graupner, G., and Pfahl, M. A retinoic acid receptor
specific element controlsthe retinoic acid receptor-ii promoter. J.
Mol. Endo., 4: 1734-1743, 1990.
39. Graupner, G., Wills, K. N., Tzukerman, M., Zhang, X-K., and
Pfahl, M.Dual regulatory role for thyroid-hormone receptors allows
control of retinoic-acid receptor activity. Nature (Lond.), 340:
653-656, 1989.
40. Elliston, J. F., Tsai, S. Y., O'Malley, B. W., and Tsai,
M-J. Superactiveestrogen receptors. J. Biol. Chem., 265:
11517-11521, 1990.
41. Klein-Hitpass, L., Schorpp, M., Wagner, U., and Ryffel, G.
U. An estrogen-responsive element derived from the 5' flanking
region of the Xenopusvitellogenin A2 gene functions in transferred
human cells. Cell, 46: 1053-1061. 1986.
42. Boylan, J. F., and Gudas, L. J. Overexpression of the
cellular retinoic acidbinding protein-l (CRABP-I) results in a
reduction in differentiation-specificgene expression in F9
teratocarcinoma cells. J. Cell Biol.. 112: 965-979,1991.
43. Crettaz, M., Baron, A., Siegenthaler, G., and Hunziker, W.
Ligand specificities of recombinant retinoic acid receptors
RAR«and RAR/1. Biochem. J.,272:391-397, 1990.
44. Clifford, J. L., Petkovich, M., Chambón, P., and Lotan, R.
Modulation byretinoids of mRNA levels for nuclear retinoic acid
receptors in murinemelanoma cells. J. Mol. Endo., 4: 1546-1555,
1990.
45. Aström,A., Pettersson, U., Krust, A., Chambón, P., and
Voorhees, J. J.Retinoic acid and synthetic analogs differentially
activate retinoic acid receptor dependent transcription. Biochem.
Biophys. Res. Commun., 173: 339-345, 1990.
46. Huang, M. E., Ye, Y. I., Chen, S. R., Chai, J. R., Lu, J-x.,
Zhoa, L., Gu, L-j., and Whang, Z-y. Use of all-frans-retinoic acid
in the treatment of acutepromyoelocytic leukemia. Blood, 72:
567-571, 1988.
4809
on June 30, 2021. © 1991 American Association for Cancer
Research. cancerres.aacrjournals.org Downloaded from
http://cancerres.aacrjournals.org/
-
1991;51:4804-4809. Cancer Res Jürgen M. Lehmann, Marcia I.
Dawson, Peter D. Hobbs, et al. Subtype-selective
ActivitiesIdentification of Retinoids with Nuclear Receptor
Updated version
http://cancerres.aacrjournals.org/content/51/18/4804
Access the most recent version of this article at:
E-mail alerts related to this article or journal.Sign up to
receive free email-alerts
Subscriptions
Reprints and
[email protected] at
To order reprints of this article or to subscribe to the
journal, contact the AACR Publications
Permissions
Rightslink site. Click on "Request Permissions" which will take
you to the Copyright Clearance Center's (CCC)
.http://cancerres.aacrjournals.org/content/51/18/4804To request
permission to re-use all or part of this article, use this link
on June 30, 2021. © 1991 American Association for Cancer
Research. cancerres.aacrjournals.org Downloaded from
http://cancerres.aacrjournals.org/content/51/18/4804http://cancerres.aacrjournals.org/cgi/alertsmailto:[email protected]://cancerres.aacrjournals.org/content/51/18/4804http://cancerres.aacrjournals.org/