From the The Verna and Marrs McLean Department of Biochemistry and Molecular Biology, Baylor College of Medicine, Houston, Texas 77030
Received for publication, October 8, 2002, and in revised form, December 5, 2002
![]() |
ABSTRACT |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
An important step in the postgenomic drug
discovery is the construction of high quality chemical libraries that
generate bioactive molecules at high rates. Here we report a cell-based
approach to composing a focused library of biologically active
compounds. A collection of bioactive non-cytotoxic chemicals was
identified from a divergent library through the effects on the
insulin-induced adipogenesis of 3T3-L1 cells, one of the most drastic
and sensitive morphological alterations in cultured mammalian cells.
The resulting focused library amply contained unique compounds with a
broad range of pharmacological effects, including glucose-uptake
enhancement, cytokine inhibition, osteogenesis stimulation, and
selective suppression of cancer cells. Adipogenesis profiling of
organic compounds generates a focused chemical library for multiple
biological effects that are seemingly unrelated to adipogenesis, just
as genetic screens with the morphology of fly eyes identify oncogenes
and neurodegenerative genes.
A complete analysis of human genome is anticipated to produce an
unprecedented number of potential drug targets. The development of high
throughput assays for these genomic pseudotargets may be a challenging
but important step for not limiting drug discovery to the "relatively
easy" targets such as G-protein-coupled receptors or particular
enzymes. An alternative or complementary effort is the construction of
high quality chemical libraries that generate bioactive molecules at
higher rates. The small size of the focused libraries would lower the
cost of screening processes and enable unique low throughput screens,
extending the scope of assays for the genomic targets and for a given
therapeutic effect.
Our approach to constructing a focused chemical library is based on the
logic of genetics. In genetic screens, clear morphological phenotypes
are often used just as a sensitive tool for discovering and analyzing
genes whose primary functions are seemingly unrelated to the
morphological phenotype. A good example is the use of eye morphology in
the fruit fly Drosophila melanogaster as a genetic tool for the analysis of genes in disease-linked signaling pathways (1). Although human diseases associated with these pathways, such as
cancer and neurodegenerative diseases, are seemingly unrelated to eye
development, the use of eye morphology as a sensitive indicator enabled
a systematic understanding of the disease-linked signaling events
(2-6). We envisioned that clear morphological phenotypes of cells
could similarly be used as a sensitive indicator of the drug effects
that are not associated directly with the morphological phenotypes.
The morphological alteration we used is the differentiation of murine
3T3-L1 fibroblasts into adipocytes, one of the most drastic and
sensitive morphological alterations in cultured mammalian cells (7). In
the presence of insulin, 3T3-L1 cells undergo differentiation into
adipocytes, which are visually distinct from the original cells because
of the presence of oil droplets in the cytoplasm (Fig. 1). The
insulin-induced adipogenesis of 3T3-L1 cells involves a number of
disease-linked proteins such as phosphatidylinositol 3-kinase,
Ras, peroxisome proliferator-activated receptor Adipogenesis Profiling--
3T3-L1 fibroblasts were plated in
96-well plates at a density of 5 × 104 cells/well and
allowed to reach maximal confluence. The confluent cells were treated
individually with 20 ng/µl of a chemical for 3 days in 100 µl of
Dulbecco's modified Eagle's medium containing of insulin (5 µg/ml)
and 10% fetal bovine serum
(FBS).1 After the removal of
insulin and the chemical, the cells were further maintained typically
for 8 days with the replacement of media every 3 days. The
effects of chemicals on the adipogenesis were evaluated under
microscope. The control wells with 1% (v/v) Me2SO
had ~5% adipocytes. The compounds that enhanced the adipogenesis >5-folds were scored to be adipogenesis-enhancing chemicals, and the
ones that completely inhibited adipogenesis without detectable toxicity
were scored to be adipogenesis-blocking chemicals. The effects of these
chemicals were confirmed multiple times by multiple laboratory members.
Cell viability was monitored by trypan blue exclusion and by
counting cell numbers.
Reverse Transcription (RT)-PCR--
Total RNA was isolated with
TRI-reagent (Molecular Research Center) at day 7 (aP2) or day 3 (osteocalcin). 5 µg of total RNA was reverse-transcribed to cDNA
by using oligo(dT) primer with avian myeloblastosis virus
reverse transcriptase for 60 min at 42 °C. The cDNA was then
amplified by using ExTaq (Takara) with following primer
pairs: 5'-AACACCGAGATTTCCTTCAA-3' and 5'-TCACGCCTTTCATAACACAT-3' for
aP2; 5'-TCTGACAAACCTTCATGTCC-3' and 5'-AAATAGTGATACCGTAGATGCG-3' for
osteocalcin. The amplification conditions are as follows: 95 °C (30 s), 60 °C (30 s), 72 °C (30 s) for 23 cycles (aP2) or 30 cycles (osteocalcin).
Glucose Uptake Study--
3T3-L1 fibroblasts were induced to
differentiate into adipocytes by incubation in a medium containing 10%
FBS, 1 µM dexamethasone, 0.5 mM
methylisobutylxanthine, and 1.7 µM insulin. After 2 days, the medium was switched to the one containing 10% FBS and 1.7 µM insulin for 2 days and then to a normal 10% FBS
medium for 3 days. After the total of 7 days, almost 100% 3T3-L1 cells
were differentiated into adipocytes. These fully differentiated cells were treated on 24-well plates with varied concentrations of chemicals (0.1% Me2SO) for 24 h and then incubated with 100 nM insulin and 2-[3H]deoxyglucose. The
cells were extensively washed, and their radioactivity was measured by
scintillation counting. All of the samples were tested in duplicate.
Cytokine Production Assay--
For the analysis of IL-6 and
TNF- Mineralization Assay--
The clonal osteoblastic cell line
MC3T3-E1, clone 14, was grown in Assays for Insulin-like Growth Factor (IGF)-activated Cancer
Cells--
The adipogenesis-blocking chemicals were assayed for their
ability to inhibit the growth of IGF-activated cancer cells. For the
discovery of inhibitors of IGF2, we used five distinct human hepatocellular carcinoma cell lines, Hep-G2, SK-Hep-1, and three lines
that we recently
characterized.2 Three of them
produce IGF2 at high levels, whereas two express ~10 times less
amounts of IGF2 as measured by ELISA, RT-PCR, and DNA microarray
experiments. Treatment with a neutralizing antibody against IGF2
selectively inhibited the growth of the IGF2-overexpressing cell lines
but had little effects on that of the cell lines with low levels of
IGF2. Thus, these cell lines served as an excellent system for
discovering the chemicals that selectively impair the growth of
IGF2-overexpressing hepatocellular carcinoma cells. For cell viability
assays, IGF2-expressing cells were plated at a density of 4 × 103 onto 96-well plates. After a 24-h incubation, the cells
were treated with varied amounts of chemicals for 72 h. The
effects of chemicals were evaluated by microscopic observation and
MTT assay. All of the samples were tested at least three times.
For reporter gene assays, IGF2-expressing cells were transfected with a
reporter construct in which a gene encoding secreted alkaline phosphatase (SEAP) is controlled by the IGF2 promoter, AP-1 sites, NFkB
sites, or the SV40 promoter. After 24 h, the transfected cells
were treated with 94G6 (0.1 µM) for 8 h. SEAP
activity was measured through fluorescence change of methylumbelliferyl
phosphate. The experiments were repeated six times. For the discovery
of inhibitors of IGF1, we used DU-145, a human androgen-independent prostate cancer cell whose growth can be stimulated by IGF1 in a
non-serum medium (13). Chemicals that inhibit the IGF1-induced growth
of DU-145 but not its serum-dependent growth were searched in the focused library of adipogenesis-blocking chemicals. DU-145 cells
were seeded onto 96-well plates at a density of 2,000 cells/well in the
presence of 1 µg/ml IGF1 or 2% FBS. After 24 h, chemicals were
added to the culture at varied concentrations. Cell proliferation was
estimated by MTT assays after 3 days. The experiments were performed in triplicate.
Adipogenesis Profiling of 10,000 Divergent Compounds--
The
divergent chemical library used for our case study was a Prime
Collection 2000 Format Q (ChemBridge). In this format, 10,000 druglike
molecules are rationally preselected to form a library that covers the
maximum pharmacore diversity with the minimum number of
compounds. Two academic groups (14, 15) have reported successful
isolations of unique compounds from a similar chemical library,
indicating that this type of chemical library contains a diverse set of
compounds that are suited for a proof-of-principle study. Our cell
morphology profiling of the 10,000-compound library identified 188 chemicals that clearly modulated the insulin-induced differentiation of
3T3-L1 cells at 20 ng/µl (Fig. 1): 81 compounds potentiated the adipogenesis; 87 compounds completely blocked
the differentiation; and 13 compounds induced other morphological
phenotypes such as adipocyte-like cells without oil droplets. Thus, the
screen reduced a pool of chemicals by 53-fold. The
adipogenesis-modulating activity of selected compounds was confirmed by
RT-PCR analysis of aP2, an adipocyte-specific fatty acid-binding
protein (an example is shown in Fig. 1E). The 188 adipogenesis-modulating chemicals that we found are apparently
non-toxic for confluent 3T3-L1 cells and almost certainly modulate
particular biologic responses in mammalian cells. The chemical
structures of adipogenesis-enhancing and -blocking compounds are
disclosed in supplementary Figs. 1 and 2.
Glucose-uptaking Insulin Sensitizers--
We first focused on the
81 chemicals that potentiated the insulin-induced adipogenesis. Their
insulin-sensitizing activity in the adipocyte differentiation suggests
that some of them enhance the insulin-induced glucose uptake with
anti-diabetic properties. This prediction was supported by the fact
that the thiazolidinedione family of anti-diabetic drugs enhances the
adipogenesis of 3T3-L1 cells through the activation of peroxisome
proliferator-activated receptor Inhibitors of Inflammatory Cytokine Production--
Recent studies
(7, 12) suggest a cross-talk between insulin-induced adipogenesis and
inflammatory responses. Anti-inflammatory drugs including
glucocorticoid, phosphodiesterase inhibitors, and salicylates stimulate
insulin-induced adipogenesis of 3T3-L1 cells, and molecular targets for
anti-inflammatory drugs such as p38, TNF- Osteogenesis Stimulators--
Insulin shares sequence homology and
biological activity with IGFs. Deficiency in IGF1, a prominent member
of IGFs, is suggested to be a cause of decrease in bone density with
aging (20, 21), and administration of IGF1 prevents the decrease of
bone density in osteoporosis patients in part by stimulating
osteogenesis (22-24). The high homology between IGF1 and insulin
suggested that the osteogenesis-enhancing activity of IGF1 may be
mimicked by the chemicals that potentiated the insulin-induced
adipogenesis. As a quick test, the adipogenesis-enhancing chemicals
were assayed for their ability to stimulate the formation of bonelike
mineral deposition in MC3T3-E1 cells. We found three compounds that
increased the mineralization at 5 µM as much as IGF1 or
ipriflavone, a clinically used anti-osteoporosis drug (Fig.
4A). Their
osteogenesis-stimulating activity was confirmed by RT-PCR analysis of
osteocalcin, a marker gene of osteoblastic differentiation. The three
compounds exhibited an increased induction of osteocalcin after 3 days
of incubation (Fig. 4B). These compounds may serve as a
small molecule tool for the mechanistic analysis of osteogenesis, and
such studies could lead to the development of pharmaceuticals for
osteoporosis, one of the most underdiagnosed and undertreated disorders
in medicine.
Suppressors of IGF-activated Cancer Cells--
We next turned our
attention to the 87 compounds that blocked the insulin-induced
adipogenesis. Both insulin and IGFs stimulate oncogenic signaling
pathways including those of Ras-MAPK and phosphatidylinositol 3-kinase-Akt, and overexpression of IGFs is often associated with cancer malignancy (25). Patients with IGF-overexpressing tumors tend to
have severe hypoglycemia despite low levels of serum insulin (known as
non-islet cell tumor hypoglycemia) (26), demonstrating a functional
overlap between oncogenic IGFs and insulin in vivo. These
considerations led to the hypothesis that the pool of the adipogenesis-blocking chemicals contains anti-cancer compounds that
suppress the IGF-stimulated survival and proliferation of malignant
tumor cells. We first examined whether the adipogenesis-blocking chemicals impair the viability of human hepatocellular carcinoma cells
that overexpress IGF2, a member of IGFs that is often produced at high
levels in liver tumors (27). We identified three chemically analogous
compounds that killed IGF2-overexpressing hepatocellular carcinoma
cells (Hep-G2) but had milder effects on the cell line with low levels
of IGF2 (SK-Hep-1) (28). Repeated experiments with three additional
human hepatocellular carcinoma cell lines that we recently
characterized2 indicated that one of the three
chemicals, 94G6, exhibited the highest cytotoxicity to IGF2-producing
hepatocellular carcinoma cells with selectivity similar to that of a
neutralizing antibody against IGF2 (Fig.
5A). This benzochromene
derivative killed the IGF2-producing cells at an IC50 of 29 nM but had ~33 times weaker effects on the hepatocellular
carcinoma cells with low level of IGF2. Reporter gene transcription
assays showed that 94G6 selectively inhibits the promoter of IGF2 in
the hepatocellular carcinoma cells, suggesting that 94G6 blocks the
autocrine loop of IGF2 (Fig. 5B). Although 94G6 may target
multiple cellular events for causing cell death, the selective
inhibition of the IGF2 autocrine loop provides a reasonable explanation
for its inhibitory effects on adipogenesis and cancer cell
survival.
Another type of IGF-associated tumors is prostate cancer, one of the
most common malignant tumors in Western countries. Elevated levels of
circulating IGF1 are strongly associated with the risk of developing
prostate cancer, and modulation of IGF1 functions by small molecules is
an attractive therapeutic approach when combined with
androgen-targeting therapies (29). For a chemical screen, we used
DU-145 androgen-independent prostate cancer cells whose growth can be
stimulated by IGF1 by as much as 2% serum. The pool of the
adipogenesis-blocking chemicals contained two analogous chemicals that
specifically inhibited the IGF1-induced growth of DU-145 cells but had
little effects on their serum-induced growth. One of them, 125B11, had
the greatest differential activity in which the simple druglike
thiazole derivative impaired the IGF1-induced growth at an
IC50 of 0.1 µM but had little effects on the
serum-dependent growth (Fig. 5C). IGF1-induced
phosphorylation of Akt and MAPK in DU-145 cells was unaffected by
125B11, suggesting that 125B11 inhibits the cell-proliferative function
of IGF1 in a way independent of the known IGF1-signaling pathway.
Deregulation of the IGF axis is associated with the initiation and
progression of many types of human carcinoma including breast (30) and
colorectal cancers (31). A focused library of adipogenesis-blocking
chemicals may serve as a source of anti-proliferative agents against
the IGF-linked cancers.
Fat cell differentiation per se has no direct link to
glucose uptake, cytokine inhibition, osteogenesis, and selective
suppression of cancer cells. Nevertheless, our proof-of-principle study
using a 10,000-compound library successfully identified non-cytotoxic bioactive compounds for these seemingly disparate pharmacological effects, just as genetics has identified non-lethal disease-linked genes by examining the eye morphology of fruit flies. We randomly picked up 70 compounds that had no detectable phenotypes in the adipogenesis profiling and assayed for their ability to modulate glucose uptake, cytokine production, IGF-selective cytotoxicity, and
osteogenesis. As expected, no significant hits were found in each
assay, indicating that the adipogenesis profiling with 3T3-L1 cells is
a good filter at least for these pharmacological effects. A data base
search revealed that one of the adipogenesis-enhancing chemicals has
been patented as an inhibitor of neuropeptide Y, a proposed attenuator
of insulin and leptin that stimulates appetite (32). Neuropeptide Y
inhibitors are expected to treat feeding disorders and heart diseases
(33). Adipogenesis profiling may find use in discovering chemicals with
such biological effects. The insulin family of hormones is involved in
many other conditions as observed in the complications of
hyperinsulinism. The insulin-linked pharmacological effects including
wound healing and anti-apoptosis (34) may be expected in
adipogenesis-modulating compounds.
One potential drawback of our approach is that the bioactive molecules
from the adipogenesis-based focused library may have side effects that
are associated with adipogenesis. However, some degree of side effects
are usually expected for any unoptimized molecules, and classical
medicinal chemistry approaches have been taken for reducing the
unwanted side effects. The high sensitivity of the morphological
transformation of 3T3-L1 cells also suggests that the
adipogenesis-modulating effects of chemicals may not necessarily be
reproduced in human. For instance, non-steroidal anti-inflammatory
drugs and phosphodiesterase inhibitors such as aspirin and caffeine are
known to enhance adipogenesis of 3T3-L1 cells but have no significant
effects on fat accumulation in human. The adipogenesis profiling is
perhaps a good filter for lead-like bioactive molecules that can be
used for further biological, chemical genetic, and medicinal chemical studies.
Adipogenesis-based profiling of more chemical compounds including
clinically proven drugs would catalog the biological activities of
small organic molecules and help to design a focused chemical library
that is small enough to be screened with unique low throughput assays
yet generates drug seeds for a broad range of disease conditions. Systematic chemical genetic studies on morphological changes of cells
could provide small molecule tools for biological studies of human
diseases as found in the role of developmental biology in the analysis
of disease-linked genes.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
, p38, or
phosphodiesterases, and known drugs for a range of diseases have been
reported to have phenotypic effects on the adipogenesis (7-12). A
morphology-based adipogenesis screen of a chemical library could
identify a pool of biologically active compounds with many distinct
pharmacological effects. Here we report a proof-of-principle study
using a library of 10,000 divergent compounds.
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
, the mouse macrophage cell line RAW264.7 was used. Cells were
seeded onto 96-well plates, and the cytokine production was induced by
adding 10 µg/ml lipopolysaccharide. Upon stimulation, each one of the
adipogenesis-enhancing chemicals was also added to the culture at
varied concentrations. After incubating for 48 h at 37 °C, the
cytokine concentrations in the culture supernatants were measured by
ELISA. For the analysis of IL-2, the mouse thymoma cell line EL-4 was
used, and the IL-2 production was induced by adding phorbol ester and
ionophore. The effects of chemicals on the IL-2 production were
similarly examined by ELISA. All of the samples were tested in triplicate.
-minimum Eagle's medium
supplemented with 10% FBS until confluent in 96-well plates. For
induction of mineralization, the cells were further incubated with 50 µg/ml ascorbic acid and 10 mM
-glycerophosphate in the
presence or absence of chemicals. On day 14, the cells were washed with
phosphate-buffered saline, fixed in 10% formalin, and washed with
distilled water. Bonelike mineral formation was evaluated by examining
the area stained by 2% (w/v) Alizarin Red S (pH 4.2).
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
View larger version (34K):
[in a new window]
Fig. 1.
Adipogenesis profiling of a library of 10,000 divergent druglike compounds. A, 3T3-L1 cells
have a morphology characteristic of fibroblasts. After chemical
treatment in the presence of insulin, the cell morphology was examined
under microscope. B, the control wells that are treated with
1% (v/v) Me2SO (DMSO) have ~5% adipocytes.
The compounds that enhanced adipogenesis >5-folds were scored to be
adipogenesis-enhancing chemicals (C), and the compounds that
completely inhibited adipogenesis without detectable cytotoxicity were
scored to be adipogenesis-blocking chemicals (D).
E, RT-PCR analysis of adipocyte-specific aP2. 3T3-L1 cells
were treated with chemicals for 3 days, and total RNA was isolated at
day 7. Typical results of four representative compounds are shown along
with the positive control of 1 µM dexamethasone
(DEX) and 0.5 mM methylisobutylxanthine
(MIX).
, a nuclear receptor that
plays an important role in adipocyte differentiation (16). In fact,
among the adipogenesis-enhancing compounds, nine had a structural
element chemically equivalent to thiazolidinedione. These known
chemicals were eliminated, and the remaining 72 chemicals were assayed
for their ability to potentiate insulin-induced glucose uptake in
cultured adipocytes. The 72 compounds contained as many as 11 molecules
that enhanced the glucose uptake at comparable levels with that of
pioglitazone, a clinically used anti-diabetic drug, demonstrating the
validness of our approach. Four of them exhibited insulin-sensitizing
activity stronger than pioglitazone at 10 µM, and the
most potent one was 124D8 (Fig. 2). Its
kinase-inhibitor-like structure is novel as an insulin sensitizer and
appears to modulate the function of insulin independently from the
major insulin pathways because 124D8 had no effects on the
phosphorylation of Akt and MAPK in 3T3-L1 cells. Adipogenesis profiling
of a larger chemical library is likely to generate a number of
glucose-uptaking compounds with a novel mechanism of action.
View larger version (26K):
[in a new window]
Fig. 2.
Identification of glucose-uptake
enhancers. Fully differentiated adipocytes were treated with 3 or
10 µM chemicals and 2-[3H]deoxyglucose in
the presence or absence of 100 nM insulin on 24-well
plates. Glucose uptake was measured by scintillation counting. The
results of the best four chemicals are shown.
, and IL-1 are involved in
adipogenesis or insulin resistance of somatic cells (11, 17-19).
Although the molecular mechanism of the cross-talk remains unclear,
these lines of evidence implicate the presence of anti-inflammatory
compounds in the pool of the adipogenesis-enhancing chemicals. We
assayed the 72 adipogenesis-enhancing chemicals for their ability to
reduce the production of three inflammatory cytokines, IL-6, IL-2, and
TNF-
. Eighteen compounds inhibited the production of a cytokine
>50% at 10 µM without notable cytotoxicity, suggesting
a high density of cytokine production inhibitors in the
adipogenesis-enhancing chemicals. Among those, the compound that we
call 69A10 inhibited the TNF-
production in macrophage RAW cells
with a IC50 of 0.3 µM (Fig.
3). A focused library of
adipogenesis-enhancing chemicals may be useful for identifying
anti-TNF-
compounds, and their mechanistic studies would clarify the
interesting cross-talk between adipogenesis and inflammatory
responses.
View larger version (15K):
[in a new window]
Fig. 3.
Inhibition of TNF-
production by 69A10. Macrophage RAW264.7 cells were seeded
onto 96-well plates, and TNF-
was induced by adding
lipopolysaccharide. Upon the stimulation, 69A10 was added to the
culture. After incubating for 48 h, the TNF-
concentrations
were measured by ELISA.
View larger version (30K):
[in a new window]
Fig. 4.
Effects of 19B8, 26E6, and 91E2 on the
osteogenesis of MC3T3-E1 cells. A, mineralization
assay. MC3T3-E1 cells were treated with 1% (v/v) Me2SO
(DMSO) or 5 µM 19B8, 26E6, or 91E2 for 14 days, and mineral deposits were stained by Alizarin Red. It is evident
that 19B8, 26E6, and 91E2 stimulate the formation of bonelike mineral
deposits. Effects of IGF1 (10 ng/ml) and ipriflavone (10 µM) are shown as a positive control. B, RT-PCR
analysis of osteocalcin. MC3T3-E1 cells were treated with chemicals for
3 days, and total RNA was isolated for RT-PCR analysis.
View larger version (19K):
[in a new window]
Fig. 5.
Discovery of anti-cancer compounds from the
adipogenesis-blocking chemicals. A,
hepatocellular carcinoma cell lines, Hep-G2 (black bars) and
SK-Hep-1 (gray bars), were treated with varied amounts of
94G6. 94G6 selectively impaired the viability of IGF2-overexpressing
Hep-G2 but had much milder effects on SK-Hep-1 with low levels of IGF2.
94G6 was as selective as a neutralizing antibody against IGF2 (100 µg/ml). The cell viability was estimated by MTT assays in triplicate.
B, specific inhibition of the IGF2 promoter by 94G6.
Hepatocellular carcinoma cells were transiently transfected with a
reporter construct in which a gene encoding SEAP is controlled by the
IGF2 promoter, AP-1 sites, B sites, or the SV40 promoter. The
transfected cells were treated with 0.1 µM 94G6 for
8 h, and SEAP activity was measured through fluorescence change of
a fluorogenic substrate. C, 125B11 inhibited the
IGF1-induced growth but not the serum-induced growth. DU-145 cells were
treated with varied amounts of 125B11 in the presence of IGF1 or 2%
fetal bovine serum (FBS).
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
![]() |
ACKNOWLEDGEMENTS |
---|
We thank M. Nakatsuka, M. Taiji, F. Nishikaku, and A. Tsuchida for assistance in assays and J. W. Harper for comments on the paper.
![]() |
FOOTNOTES |
---|
* This work was supported in part by U. S. Department of Defense Prostate Cancer Research Program.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.
The on-line version of this article (available at
http://www.jbc.org) contains Figs. 1 and 2.
Predoctoral fellow of the U. S. Department of Defense.
§ These authors contributed equally to the work.
¶ To whom correspondence should be addressed. E-mail: muesugi@ bcm.tmc.edu.
Published, JBC Papers in Press, December 19, 2002, DOI 10.1074/jbc.M210283200
2 K. Murakami and M. Uesugi, unpublished data.
![]() |
ABBREVIATIONS |
---|
The abbreviations used are: FBS, fetal bovine serum; RT, reverse transcription; IL, interleukin; TNF, tumor necrosis factor; ELISA, enzyme-linked immunosorbent assay; IGF, insulin-like growth factor; SEAP, secreted alkaline phosphatase; AP, activating protein; MAPK, mitogen-activated protein kinase; MTT, 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide.
![]() |
REFERENCES |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
1. | Thomas, B. J., and Wassarman, D. A. (1999) Trends Genet. 15, 184-190[CrossRef][Medline] [Order article via Infotrieve] |
2. | Wassarman, D. A., Therrien, M., and Rubin, G. M. (1995) Curr. Opin. Genet. Dev. 5, 44-50[Medline] [Order article via Infotrieve] |
3. | Luo, H., and Dearolf, C. R. (2001) Bioessays 23, 1138-1147[CrossRef][Medline] [Order article via Infotrieve] |
4. | McCall, K., and Steller, H. (1997) Trends Genet. 13, 222-226[CrossRef][Medline] [Order article via Infotrieve] |
5. | Burke, R., and Basler, K. (1997) Curr. Opin. Neurobiol. 7, 55-61[CrossRef][Medline] [Order article via Infotrieve] |
6. |
Min, K. T.,
and Benzer, S.
(1999)
Science
284,
1985-1988 |
7. | Rosen, E. D., and Spiegelman, B. M. (2000) Annu. Rev. Cell Dev. Biol. 16, 145-171[CrossRef][Medline] [Order article via Infotrieve] |
8. |
Klemm, D. J.,
Leitner, J. W.,
Watson, P.,
Nesterova, A.,
Reusch, J. E.,
Goalstone, M. L.,
and Draznin, B.
(2001)
J. Biol. Chem.
276,
28430-28435 |
9. |
Ho, I. C.,
Kim, J. H.,
Rooney, J. W.,
Spiegelman, B. M.,
and Glimcher, L. H.
(1998)
Proc. Natl. Acad. Sci. U. S. A.
95,
15537-15541 |
10. |
Dowell, P.,
Flexner, C.,
Kwiterovich, P. O.,
and Lane, M. D.
(2000)
J. Biol. Chem.
275,
41325-41332 |
11. |
Engelman, J. A.,
Lisanti, M. P.,
and Scherer, P. E.
(1998)
J. Biol. Chem.
273,
32111-32120 |
12. |
Engelman, J. A.,
Berg, A. H.,
Lewis, R. Y.,
Lin, A.,
Lisanti, M. P.,
and Scherer, P. E.
(1999)
J. Biol. Chem.
274,
35630-35638 |
13. | Iwamura, M., Sluss, P. M., Casamento, J. B., and Cockett, A. T. (1993) Prostate 22, 243-252[Medline] [Order article via Infotrieve] |
14. |
Komarov, P. G.,
Komarova, E. A.,
Kondratov, R. V.,
Christov-Tselkov, K.,
Coon, J. S.,
Chernov, M. V.,
and Gudkov, A. V.
(1999)
Science
285,
1733-1737 |
15. |
Mayer, T. U.,
Kapoor, T. M.,
Haggarty, S. J.,
King, R. W.,
Schreiber, S. L.,
and Mitchison, T. J.
(1999)
Science
286,
971-974 |
16. |
Lehmann, J. M.,
Moore, L. B.,
Smith-Oliver, T. A.,
Wilkison, W. O.,
Willson, T. M.,
and Kliewer, S. A.
(1995)
J. Biol. Chem.
270,
12953-12956 |
17. | Ohsumi, J., Sakakibara, S., Yamaguchi, J., Miyadai, K., Yoshioka, S., Fujiwara, T., Horikoshi, H., and Serizawa, N. (1994) Endocrinology 135, 2279-2282[Abstract] |
18. | Petruschke, T., and Hauner, H. (1993) J. Clin. Endocrinol. Metab. 76, 742-747[Abstract] |
19. | Zick, Y. (2001) Trends Cell Biol. 11, 437-441[CrossRef][Medline] [Order article via Infotrieve] |
20. | Rosen, C. J., and Donahue, L. R. (1998) Proc. Soc. Exp. Biol. Med. 219, 1-7[Abstract] |
21. | Baker, J., Liu, J. P., Robertson, E. J., and Efstratiadis, A. (1993) Cell 75, 73-82[Medline] [Order article via Infotrieve] |
22. | Bianda, T., Hussain, M. A., Glatz, Y., Bouillon, R., Froesch, E. R., and Schmid, C. (1997) J. Intern. Med. 241, 143-150[CrossRef][Medline] [Order article via Infotrieve] |
23. | Ebeling, P. R., Jones, J. D., O'Fallon, W. M., Janes, C. H., and Riggs, B. L. (1993) J. Clin. Endocrinol. Metab. 77, 1384-1387[Abstract] |
24. | Grinspoon, S., Baum, H., Lee, K., Anderson, E., Herzog, D., and Klibanski, A. (1996) J. Clin. Endocrinol. Metab. 81, 3864-3870[Abstract] |
25. |
Yu, H.,
and Rohan, T.
(2000)
J. Natl. Cancer. Inst.
92,
1472-1489 |
26. | Daughaday, W. H. (1995) Diabetes Rev. 3, 62-72 |
27. |
Scharf, J. G.,
Dombrowski, F.,
and Ramadori, G.
(2001)
Mol. Pathol.
54,
138-144 |
28. | Zvibel, I., Halay, E., and Reid, L. M. (1991) Mol. Cell. Biol. 11, 108-116[Medline] [Order article via Infotrieve] |
29. | Djavan, B., Waldert, M., Seitz, C., and Marberger, M. (2001) World J. Urol. 19, 225-233[CrossRef][Medline] [Order article via Infotrieve] |
30. |
Sachdev, D.,
and Yee, D.
(2001)
Endocr. Relat. Cancer
8,
197-209 |
31. |
Hassan, A. B.,
and Macaulay, V. M.
(2002)
Ann. Oncol.
13,
349-356 |
32. | Deleted in proof |
33. | Balasubramaniam, A. (2002) Am. J. Surg. 183, 430-434[CrossRef][Medline] [Order article via Infotrieve] |
34. |
Dore, S.,
Kar, S.,
and Quirion, R.
(1997)
Proc. Natl. Acad. Sci. U. S. A.
94,
4772-4777 |
|
HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
All ASBMB Journals | Molecular and Cellular Proteomics |
Journal of Lipid Research | Biochemistry and Molecular Biology Education |