From the Advances in the understanding of the retinoid
signaling mechanism has allowed the discovery of highly selective
retinoids that activate only one specific receptor class, subtype, or
signaling pathway. These novel compounds lack certain of the common
retinoid toxicities and therefore suggest promising new approaches for therapeutic applications. We describe here a new compound,
6-[3-(1-adamantyl)-4-hydroxyphenyl]-2-naphthalene carboxylic acid
methyl ester (MX84), that is selectively activated in macrophages,
leading to killing of only macrophage monocyte type cells in
vitro. We provide evidence that MX84 is an inactive precursor
that is converted into an active apoptosis-inducing retinoid in
macrophages. The macrophage activity is also secreted, and our data
suggest that the secreted activity is a phospholipase D type activity.
Our observation may lead to the development of molecules that are
highly macrophage-selective apoptosis inducers in vivo and
that could represent important novel therapeutics against diseases
caused by excessive macrophage activity.
Retinoids exert their multiple biological effects through
interaction with specific nuclear proteins, the retinoid receptors. These regulatory proteins belong to a large group of transcription factors, the steroid/thyroid hormone nuclear receptor superfamily. Two
classes of retinoid receptors have been identified, the
RARs1 and RXRs, each of which
has three subtypes, Although natural retinoids are currently used in the treatment of
certain skin diseases and in the treatment of acute promyelocytic leukemia (2-3), their medical applications are restricted by multiple
side effects, caused by the intimate and universal role of retinoids in
regulation of gene expression, orchestrating cell proliferation,
differentiation, and embryonic development (4-6). This broad activity
of the natural retinoids is due to their fairly indiscriminate binding
to and activation of the various receptor subtypes. Thus,
all-trans-retinoic acid (tRA) activates all RAR subtypes,
whereas 9-cis-RA activates all RARs and, in addition, the
RXRs. The more recent findings that some synthetic retinoids exert
their biological effects by selective binding or activating only
specific RAR subtypes (either In addition to receptor-selective compounds, synthetic retinoids could
also be found to function in some of the retinoid signaling pathways
but not in others. For instance, anti-AP-1-selective compounds show
transcriptional repression but not transcriptional activation
activities (16). These types of compounds lack many of the typical
retinoid activities, and thus toxicities, and may find applications as
specific inhibitors of certain disease-associated genes, activated by
the transcription factor activator protein-I. Another interesting class
of novel retinoids has been recently identified that induce apoptosis
(17-19, 22). These molecules have been shown to specifically bind or
transactivate RAR Using the same cell-based antiproliferation/cell killing screen, we
also discovered one molecule, MX84, that induced cell death in
monocyte/macrophage-derived cell lines (mMn1, mMn2, and mMn3) but was
inactive against more than 30 other cell lines, including solid
tumor-derived cell lines. We show here that MX84 is inactive as a
transcriptional activator in CV-1 cells but at the same concentration
can activate gene transcription in macrophage type cells.
Interestingly, in these latter cells, the compound is converted from an
inactive retinoid precursor into an active retinoid that induces
apoptosis. The monocyte/macrophage type cell lines investigated here
were also found to secrete a protein into the medium that allows the
activation of the retinoid. This cell-specific enzymatic conversion of
an inactive retinoid precursor into an apoptosis-inducing compound may
be exploited as a scheme for the developing of macrophage cell
type-selective retinoids with apoptotic and/or other desirable
activities.
Cell Culture, Transfections, and MTT Test--
CV-1, H661,
J774A.1, LNCaP, and RAW264.1 cell lines used in this study were
maintained in the recommended growth medium, either Dulbecco's
modified Eagle's medium or RPMI 1640 medium (Irvine Scientific),
supplemented with fetal calf serum (Irvine) and penicillin and
streptomycin (Life Technologies, Inc.).
Sidney Kimmel Cancer Center, ¶ Galderma
Research,
Maxia Pharmaceuticals, Inc.,
San Diego, California 92121
ABSTRACT
Top
Abstract
Introduction
Procedures
Results
Discussion
References
INTRODUCTION
Top
Abstract
Introduction
Procedures
Results
Discussion
References
,
, and
, and several isoforms. Cell type-
and developmental stage-specific expression patterns of these receptor
subtypes and isoforms are believed to contribute to distinct cell type
and developmental stage gene expression patterns that are regulated by
retinoids. Further amplifying the diversity of retinoid-regulated gene
expression patterns, these receptors bind as RXR/RAR heterodimers or
RXR homodimers to specific DNA sequences in the promoter region of retinoid-responsive genes or interact with other transcription factors,
such as activator protein-I (reviewed in Ref. 1).
,
, or
) (7-10) or only RXRs
(11-14) were encouraging. The first RAR subtype-selective compound,
Adapalene, an RAR
/RAR
-selective retinoid, has now been approved
in many countries for the topical treatment of acne, showing fewer side
effects in this application than tRA. RXR-selective compounds were
recently found to be effective in animal models for the treatment of
type II diabetes (15).
(20-22). The first apoptosis-inducing retinoids
described,
N-(4-hydroxyphenyl)-all-trans-retinamide and
CD437 (Refs. 23-26 and 27-29, respectively), are, however,
relatively indiscriminate apoptosis inducers and are thus very toxic
in vivo at effective
concentrations.2 However,
some of us showed recently that new apoptosis-inducing retinoids can be
identified that show cell type selectivity (22). Importantly, one of
the molecules identified by an in vitro screen was effective
against a human non-small cell lung cancer xenograft in
vivo, at concentrations at which it did not induce major side effects (22).
EXPERIMENTAL PROCEDURES
Top
Abstract
Introduction
Procedures
Results
Discussion
References
-galactosidase
activities.
Apoptotic Cell Death Assay--
Apoptotic cell death detection
was performed using the Cellular DNA Fragmentation ELISA kit
(Boehringer Mannheim), essentially as recommended by the manufacturer.
3 × 104 RAW 264.1 cells/well were used for the assay.
RAW 264.1 cells were exposed for 15 h to either 106
M MX84 or 10
6 M CD437.
10
6 M staurosporine, a non-retinoid inducer
of apoptosis, was used as a positive control. Peptide fluoromethyl
ketones ZVAD-fmk and ZDEVD-fmk were obtained from Enzyme Systems
Products and were applied at 50 µM concentrations
simultaneously with the retinoids.
Thin-layer Chromatography--
Confluent flasks of J774A.1 and
RAW264.1 cells were incubated for 18 h in 5 ml of growth medium
(Dulbecco's modified Eagle's medium and RPMI 1640, respectively) in
the presence of 106 M MX84. The growth medium
was then collected, floating cells were removed by centrifuging, and 2 ml of methanol/methylene chloride (10:90) were added. After vigorous
vortexing, the organic phase was separated by centrifugation, and 0.8 ml of the organic phase was concentrated to 10 µl using a Speed Vac
SC100 (Savant Instruments, Inc.) and analyzed by TLC. For the assay, 1 µl of the concentrate was loaded onto 2 × 7-cm TLC plate silica
gel 60 (EM Separations Technology, Gibbstown, NJ) with a
preconcentration zone. As a running solvent, methanol/methylene
chloride (10:90) mix was used. After a run of 3 min, the plates were
analyzed with a Shimadzu CS-9301 PC dual wavelength flying spot
scanning densitometer.
Enzymatic Assays and Inhibition Studies-- To obtain a concentrated solution of the macrophage-secreted high molecular weight activation enzyme(s) (converting MX84 into an active compound), RAW264.1 cells were washed three times with PBS and then incubated in PBS for 6 h at 37 °C, 6% CO2. The PBS was collected, floating cells were removed by spinning, and the supernatant was concentrated using an Ultrafree-15 Centrifugal Filter Device, Biomax-100K NMWL membrane (Millipore Corp., Bedford, MA).
All commercially available enzymes used in this study were purchased from Sigma. Lipase from human pancreas (6 units), lipase from Candida rugosa (50-1000 units), and esterase from porcine liver (4-20 units) were tested for esterase activity toward MX84 as follows: the enzymes were incubated for 20 h at 37 °C in 0.2 ml of PBS containing 5 × 10 ![]() |
RESULTS |
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Identification of a Macrophage-selective Retinoid
In the last few years, novel selective retinoids and
retinoid-related molecules have been identified that show potential for medical applications. A particularly interesting class of retinoids is
molecules that induce apoptosis. However, the first such molecules described appear to induce apoptosis fairly
indiscriminately.2 Recently, we were able to demonstrate
that selective apoptosis inducers with good effectivity/toxicity
profiles in vivo could be identified (22). Using a high
throughput biological activity assay, we analyzed more than a thousand
retinoids for their ability to inhibit cell growth or induce cell
killing on a large panel of cell types (22). In brief, cell cultures
were incubated with 2 × 106 M
concentrations of the various retinoids for periods of 5 days, after
which the percentages of live cells were determined using a
colorimetric (MTT) assay. Among more than 1000 retinoids analyzed, we
found one compound, MX84, that under these conditions effectively killed macrophage/monocyte type cells but did not kill more than 40 other cell lines investigated. An excerpt of the screening results is
shown in Fig. 1, in which MX84 is
compared with the strong apoptosis inducer CD437 and the natural
retinoid tRA. As can be seen, CD437 indiscriminately induces cell
growth arrest/cell killing in all cell types shown, whereas MX84 is
active only in monocyte/macrophage type cell lines J774A.1 and RAW264.1
under the conditions used. tRA showed a significant growth inhibitory effect only on few of the cell lines, including F-9 cells, as well as
HL-60 cells. In these cell types, tRA is known to induce differentiation, which includes cell growth arrest. Interestingly, MX84
appears not to be a differentiation-inducing agent because it shows no
activity in F-9 cells and PC19 cells. To verify the screening results,
titration experiments were carried out comparing the growth
inhibitory/cell killing effects of MX84 and tRA at various
concentrations on several cell lines. As can be seen (Fig. 2, a and b) at
10
6 M, MX84 is a very effective inhibitor of
two monocyte/macrophage cell lines J774A.1 and RAW264.1 but not of the
non-small cell human lung cancer cell line H661 or the prostate cancer
cell line LNCaP (Fig. 2, c and d, respectively).
On this latter cell line, tRA showed a weak inhibitory effect at
10
6 M, consistent with published results,
whereas MX84 did not. Thus, under the in vitro conditions
used, MX84 is a selective inhibitor of monocyte/macrophage type cell
lines.
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MX84 Induces Apoptosis in Macrophage Type Cells
To characterize the mode of action of this new retinoid in
macrophages, we applied 106 M concentrations
of either MX84 or the apoptosis-inducing retinoid CD437 to RAW 264.1 cells. After incubation with MX84, the macrophage cultures demonstrated
morphological changes characteristic of cells undergoing apoptosis
(data not shown), such as formation of membrane-bound vesicles and cell
shrinkage due to condensation of cytoplasm; these were similar to the
changes observed in cells treated with CD437 or with staurosporine,
which are known inducers of apoptosis. Furthermore, using a cellular
DNA fragmentation ELISA kit (Boehringer Mannheim), we found that MX84
induced apoptosis in these cells almost as efficiently as CD437 (Fig.
3), whereas tRA showed minimum basal
activity (not shown).
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Peptide fluoromethyl ketones ZVAD-fmk, an inhibitor of caspases with selectivity toward caspase 1 (32), and ZDEVD-fmk, a specific inhibitor of caspase 3, completely protected the macrophages from MX84-induced or CD437-induced apoptotic cell death (data not shown). We have previously shown that retinoid-induced apoptosis/cell killing can be inhibited by specific caspase inhibitors (38). Thus, our observation here that the antimacrophage activity of MX84 is inhibited by caspase inhibitors provides further proof that MX84 is an apoptosis-inducing retinoid in macrophages.
Cell Type-specific Conversion of MX84 into a Transcriptional Activator
It is generally believed that retinoids require a carboxyl
terminus to efficiently bind and transactivate their nuclear receptors. Consistent with this, we observed that MX84 was very inefficient in
activating a retinoid-responsive reporter gene in CV-1 cells, when
compared to tRA, a cell line commonly used to measure transactivation capacities of nuclear receptor ligands. In contrast, CD437, known to be
a potent transcriptional activator (39), induced the
retinoid-responsive reporter gene expression even more strongly than
tRA when applied at same concentration in CV-1 cells (See Fig.
4a). When we carried out
transactivation studies with the macrophage type cell line RAW264.1, we
observed that MX84 behaved similarly to CD437, leading to induction of
a retinoid-responsive reporter gene in the presence of RAR. Thus,
although MX84 is inactive in CV-1 cells in terms of induction of
apoptosis and in functioning as a transcriptional activator, it gains
both functions in macrophage type cells (Fig. 4b).
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Conversion of MX84 into an Active Retinoid
At least two different hypotheses appear reasonable to explain the cell type-specific activity of MX84: (i) the retinoid signaling machinery differs in these cells from most other cell types, containing cofactors/coactivators that allow MX84 to be active, or (ii) MX84 is enzymatically converted in these cells into an active compound. In Fig. 5, the formulas of tRA, MX84, and CD437 are compared. It is apparent that the methyl ester MX84 could be converted into a free acid end compound. This free acid would in fact be identical to the apoptosis-inducing retinoid CD437, which functions in macrophage type cells as an inducer of apoptosis and as a transcriptional activator.
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To examine this hypothesis, we incubated 106
M MX84 overnight with macrophage cell lines. When lysates
of these cells were subsequently analyzed by thin-layer chromatography
(TLC), we observed that a large portion of MX84 was converted to a
compound migrating identical to CD437 on TLC plates (Fig.
6). In contrast, incubation of MX84 with
the non-macrophage cell line CV-1 resulted in very little conversion,
and no conversion was seen with H661 cells (not shown).
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To investigate whether the MX84 conversion could be associated with
secretory functions of macrophage type cells, we added 106 M MX84 to "macrophage-conditioned"
medium, i.e. medium in which the macrophage cell lines had
been grown overnight. Both macrophage cell lines, J774A.1 and RAW
264.1, were found to secrete the MX84 conversion activity into the
medium (Fig. 6). Thus, MX84 is activated in macrophage type cells, and
in addition, this activating activity is secreted by these cells.
Specificity of the Macrophage Activation Enzyme(s)
We observed that only macrophage/monocyte type cells excreted an MX84 activation activity into the growth medium. We took advantage of this observation to analyze the specificity of the excreted "esterase" activity in some detail. This was of interest because defining the selectivity of the MX84 converting/activating activity should allow the future design of compounds that are most selectively activated by macrophages in vivo. In this respect, it is important to note that a considerable variety of enzymes contain or can be expected to contain "esterase" activities, which would allow for the conversion of the MX84 (a methyl ester) into a carboxylic acid. Potential converting enzymes include certain proteases and phospholipases, as well as cholesterol esterases and lipases. To test for the presence of these enzymes, we analyzed MX84 conversion in RAW264.1 supernatants. For this, the cells were cultured for 6 h in PBS, after which the secreted proteins were concentrated using an Ultrafree-15 centrifuge device.
Protease Inhibitors-- Use of known effective concentrations of various protease inhibitors (Table I) revealed that the activity that converts MX84 in RAW 264.1 cells could be completely inhibited by the thiol-blocking agents pCMPS and DTNB. The activity was also inhibited by the amino peptidase inhibitor bestatin, by the chymotrypsin inhibitor chymostatin, and by the serine protease inhibitor Pefabloc SC, suggesting a process involving the conversion of protein precursor to the active form of protein. No inhibition of the esterase activity was observed either with effective concentrations of several other inhibitors of serine, cysteine, and aspartate proteases, metalloendopeptidases, and metalloproteases, nor with turkey egg white trypsin inhibitor. Sodium fluoride, even at the highest concentration tested (10 mM), was unable to inhibit the esterase activity, as well as the C-1 esterase inhibitor and PMSF, (unless very high concentrations of the latter were used (33 and 57% inhibition by 3 and 10 mM PMSF, respectively). The esterase activity was also not inhibited by the caspase inhibitors ZVAD-fmk and ZDEVD-fmk. Overall, these data suggest that a cysteine residue is more likely to be part of the active site than a serine residue. In addition, because certain proteases, such as cysteine and serine protein inhibitors, did not inhibit the conversion of MX84, proteases appear to be unlikely candidates.
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Phospholipase Inhibitors-- To evaluate possible phospholipase involvement in MX84 activation in macrophages, specific phospholipase inhibitors were tested. Data obtained with several concentrations of specific phospholipase inhibitors are summarized in Table II. The conversion activity was not inhibited by 100 nM MAFP, an inhibitor that is specific for the calcium-dependent phospholipase A2 (PLA2), or by 100 nM HELSS, which is specific for the calcium-independent PLA2. Only when these compounds were applied at 1 µM concentrations could some inhibition be observed. The activity was affected by the PLD inhibitor 1,10-phenanthroline; however, it was not inhibited by 2,3-diphosphoglycerate, another known PLD inhibitor. In contrast, the antitumor drug suramin, which has been reported to inhibit PLD activity (32), was found to inhibit the macrophage activity in a dose-dependent manner. The antitumor ether lipid sn-ET-18-OCH3, which has also been reported to interfere with PLD activity (35, 36), was also a potent inhibitor of PLC. The aminosteroid U73122, an inhibitor of PLA2, PLC, and PLD (32, 34), also suppressed the conversion of MX84 into CD437. The antiviral and antitumoral xanthogenate compound D609, known as an inhibitor of PLC and PLD (32, 33), was effective only at highest concentrations tested, also suggesting the possible involvement of a PLD-like activity.
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DISCUSSION |
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With few exceptions, the cell lines studied (80-90% of cell
lines) were found to be sensitive to 106 M
concentrations of the RAR
-selective, apoptosis-inducing retinoid CD437. In contrast, only monocyte/macrophage cell lines were sensitive to 10
6 M of MX84. In addition to the
important role of these cell types in the immunological defense,
excessive macrophage activity has been associated with a number of
widespread diseases such as arthritis, multiple sclerosis, septic
shock, and so forth. Control of macrophage populations could therefore
provide an important approach for the treatment and management of those
diseases. We describe here a compound, MX84, that from among more than
1000 compounds tested showed highly selective antimacrophage activity
in vitro. We analyzed the mechanisms by which MX84 can exert
this highly selective killing activity. We observed that in macrophage
type cells, MX84 behaves as a transcriptionally active retinoid, but it
is inactive in other cell types, such as CV-1 cells. In addition, MX84
induces apoptosis in macrophages. Chemical analysis of macrophage
extracts revealed that MX84 is converted into a compound that shows
migrations on TLC plates identical to those of the apoptosis-inducing
compound CD437. This observed conversion is not by itself that
surprising, because chemically, MX84 is a methyl ester of CD437 (see
Fig. 5). What is surprising is the high degree of cell type selectivity for this conversion observed in vitro. The high degree of
selectivity, however, is not expected to hold in vivo,
because esterases are quite ubiquitous (consistent with our preliminary
in vivo results). To eventually be able to obtain a compound
that is selectively active against macrophage type cells in
vivo, it is not only important to analyze the mechanism by which
MX84 exerts its activity in these cells, but also to determine the
nature of the MX84 activating enzymes. Our observation that all
macrophage type cell lines secreted the specific activity may be of
importance and is consistent with previous reports and the known
characteristics of these cell types.
Our inhibition studies revealed that the macrophage-secreted esterase activity was completely inhibited by thiol-blocking agents, such as pCMPS and DTNB. In addition, the enzymatic activity was inhibited in part by such agents as the amino peptidase inhibitor bestatin, the chymotrypsin inhibitor chymostatin, and the serine protease inhibitor Pefabloc SC (Table I). PMSF, however, did not inhibit the activity (unless used at very high concentrations). Taken together, these data suggest the possible involvement of a cysteine residue(s) in a single active site, and, less likely, the involvement of a certain serine residue(s). Noteworthy, however, is that no inhibition of the esterase activity was observed with several other inhibitors for cysteine and serine proteases, such as antipain-dihydrochloride, aprotinin, E-64, leupeptin, and turkey egg white trypsin inhibitor, as well as with peptide inhibitors for caspases.
Studies with commercially available enzymes indicate that hydrolases of several different classes could be involved in the MX84 ester bond cleavage. These enzymes include phospholipases, chymotrypsin-like esterases, or lipases. These results are consistent with the observation that PLD activity was seen in J774 cells, RAW264 cells, and in all other myeloid cell lines. In addition, constitutive secretion of the PLD activity into the medium by J774 cells has been reported (40). Not unexpectedly, our preliminary studies show that three additional monocyte/macrophage cell lines tested (WEHI-3, WEHI-265, and P388) secreted MX84 conversion activity.
The activity in RAW264.1 macrophages was inhibited by the PLD inhibitor 1,10-phenanthroline in a dose-dependent manner. The antitumor drug suramin, previously reported to inhibit PLD activity (32), also demonstrated dose-dependent inhibition of the macrophage-associated esterase activity, as did an antiviral and antitumoral xanthogenate compound D609, known as an inhibitor of PLC and PLD (32, 33), and an aminosteroid U73122, an inhibitor of PLA2, PLC, and PLD (32, 34). The antitumor ether lipid sn-ET-18-OCH3, a potent inhibitor of PLC that is also reported to interfere with PLD activity (35, 36), was also found to be an inhibitor of the RAW264.1 macrophage-associated esterase activity. However, the MX84 ester bond cleavage was not inhibited in our experiments by PLD inhibitor 2,3-diphosphoglycerate. This may suggest a highly specific phospholipase as the MX84 converter.
To further test this hypothesis, we tested whether the naturally
occurring substrates for phospholipases,
L--phosphatidylcholine, and
L-
-lysophosphatidylcholine, could function, when in
excess, as inhibitors of the MX84 ester bond cleavage. Indeed, we found that both L-
-phosphatidylcholine and
L-
-lysophosphatidylcholine, when present at a ~10-fold
molar excess over MX84, were efficient inhibitors of the conversion,
with L-
-phosphatidylcholine being the more potent
inhibitor. In contrast, these two substrates were unable to inhibit the
esterase from porcine liver under similar conditions. Thus, these data
are consistent with and support the hypothesis that the methyl ester
MX84 is converted into CD437 by an esterase activity of a
macrophage-secreted phospholipase.
Expression of a 100-kDa phospholipase A2 (PLA2) has been reported for several macrophage cell lines, including J774 (41) and RAW264 (42). Both calcium-independent and calcium-dependent activities of high molecular weight PLA2 in the macrophages were described (43-46), as well as Ca2+-independent lysophospholipase activity of the enzyme purified from the RAW264.7 cells (42). However, in our hands, phospholipases A2 from different sources did not possess MX84 conversion activity. Consistent with this is our observation that the conversion activity was not affected either by HELSS or by MAFP, inhibitors for calcium-independent and calcium-dependent phospholipases A2, respectively (unless very high concentrations of these PLA2 inhibitors were used).
Thus, our analyses provide certain clues to the nature of the MX84 conversion enzyme that can now be tested in vitro and in vivo by designing other MX84 derivatives. Possibly our most important observation, however, is that along with an extensive search for receptor subtype-specific, cell-specific, and apoptosis-inducing retinoids, the design of inactive retinoid precursors that can be activated by cell type-specific enzymes can be used as an alternative strategy to develop effective and cell type-specific retinoids for the treatment of diseases such as inflammation and cancer.
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FOOTNOTES |
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* This work was supported by Grant CA55681 from the National Institutes of Health (to M. P.).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
§ Supported by a postdoctoral fellowship from Galderma Research, Inc.
** To whom correspondence should be addressed: Sidney Kimmel Cancer Center, 10835 Altman Row, San Diego, CA 92121. Tel.: 619-623-9632; Fax: 619-824-1967.
The abbreviations used are: RAR, retinoic acid receptor; RXR, retinoid X receptor; CAT, chloramphenicol acetyltransferase; MX84, 6-(3-(1-adamantyl)-4-hydroxyphenyl)-2-naphthalene carboxylic acid methyl ester; CD437, 6-(3-(1-adamantyl)-4-hydroxyphenyl)-2-naphthalene carboxylic acid; pCMPS, p-chloromercuriphenylsulfonic acidDTNB, 5,5'-dithio-bis(2-nitrobenzoic acid)HELSS (haloenol lactone suicide substrate), E-6-(bromomethylene)-tetrahydro-3-(1-naphthalenyl)-2H-pyran-2-oneMAFP, methylarachidonyl fluorophosphonateMES, 2-(N-morpholino)ethanesulfonic acidPBS, phosphate-buffered salinePMSF, phenylmethylsulfonyl fluoridetRA, all-trans-retinoic acidTREpal, thyroid hormone-responsive palindromic elementMTT, 3-(4,5-dimethyl thiazol-2-yl)-2,5-diphenyl tetrazoliumELISA, enzyme-linked immunosorbent assayPLD, phospholipase DPLC, phospholipase C.
2 X.-P. Lu and M. Pfahl, unpublished results.
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REFERENCES |
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