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Experimental Therapeutics |
Department of Nuclear Receptor Discovery, Ligand Pharmaceuticals Inc., San Diego, California 92121
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ABSTRACT |
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 Top ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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(PPAR), and
lipoprotein lipase was elevated in LGD1069-responding tumors. Further
analysis showed that gene expression changes occurred rapidly, in as
little as 6 h, after the first dose of LGD1069.
Immunohistochemical analysis showed that aP2 protein was also highly
expressed in responding tumors when compared with control or
nonresponding tumors. More importantly, aP2 protein was localized in
the tumor cells in addition to the adipocytes present in the tumors.
Similar changes in gene expression and inhibition in growth were seen
in tumor cells (cloned from
N-nitroso-N-methylurea-induced carcinoma)
exposed to LGD1069 in vitro. These data suggest that
tumor regression by LGD1069 involves differentiation induction along
the adipocyte lineage. |
INTRODUCTION |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Recently, we have demonstrated that LGD1069 (a selective ligand for the RXRs; Ref. 7 ) caused complete regression in 72% of NMU-induced rat mammary carcinomas (8) . More importantly, LGD1069 also caused complete regression of tumors that previously failed TAM therapy (9) . LGD1069 also has efficacy as a chemopreventive agent in the NMU-induced rat mammary carcinoma model that is equivalent to that of TAM (10) . Mechanistically, LGD1069 causes regression of rat mammary tumors by inhibiting tumor cell proliferation (8) . LGD1069 in vivo can also interfere with estrogen- or TAM-induced proliferation in the uterus (10) . We have also observed in vitro that the RXR ligand LGD1069 inhibits the growth of human breast cancer cells and interferes with estrogen signaling and induces lipid accumulation in these cells.5
RXR ligands including LGD1069 have been shown to induce
adipocyte-specific gene expression in preadipocytes and in human
liposarcoma cells (Refs. 11
, 12
and personal
observation6
). The expression of adipocyte-specific genes may be important in
certain cancers, because an increase in aP2, adipsin and PPAR
expression levels have been correlated with the inhibition of cellular
proliferation in tumors of liposarcoma patients (13)
. In
addition, the loss of aP2 expression has been shown to be associated
with the progression of human bladder transitional cell carcinoma
(14
, 15)
. Therefore, to evaluate the effect of LGD1069 at
the molecular level in responding rat mammary carcinomas, the
expression of the adipocyte-related genes aP2,
adipsin, and PPAR, and of genes encoding
enzymes involved in fatty acid uptake and synthesis, including
LPL (16)
, SCD1 (17)
,
and FAS (18)
was examined. A significant
increase in expression of the adipocyte-related genes was observed in
responding tumors when compared with control tumors or tumors that had
failed LGD1069 therapy (nonresponders). Further analysis showed that
aP2 protein was also highly expressed in responding tumors when
compared with control or nonresponding tumors. Interestingly, aP2
protein was localized in the tumor cells in addition to the adipocytes
present in the tumors. Similar changes in gene expression and
inhibition in growth were seen in NMU417 cells (tumor cells isolated
and cloned from an NMU-induced rat mammary tumor) exposed to LGD1069
in vitro. Hence, induction of adipocyte-related gene
expression correlated with the regression of NMU-induced rat mammary
carcinomas by LGD1069 treatment. Thus, tumor regression by LGD1069
involves differentiation induction along the adipocyte lineage.
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MATERIALS AND METHODS |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Tumor Induction, Measurement, and Scoring.
The procedures for tumor induction and their measurement and scoring in
the mammary gland of female Sprague Dawley rats were described earlier
(8)
. In the present study, tumors were allowed to grow to
a size of 200 mm2 in cross-sectional area prior
to initiating oral administration of LGD1069. Animals were administered
LGD1069, suspended in an aqueous solution composed of 10% (v/v)
polyethylene glycol (Mr
400)/Tween 80 (99.5:0.5) and 90% of 1% (w/v) carboxy-
methylcellulose (Sigma Chemical Co., St. Louis, MO) at 100 mg/kg
(p.o., 7 days a week), and the treatment continued until
responding tumors showed at least two consecutive decreases in area
totaling >40% from their maximum size. Progressively growing tumors
(nonresponding tumors), grew over the course of treatment (their final
area was at least 40% greater than their initial area) and were used
to evaluate gene expression differences between the tumors that
responded and the tumors that did not respond to LGD1069 treatment.
These studies have a treatment period in the range of 20–60 days, and
the growth of all of the tumors was monitored twice weekly throughout
the course of the study. The last dose of LGD1069 was given 6 h
prior to the killing of the animals. For statistical analysis of
differences in gene expression levels, studies were performed using
nine tumors of each category: control tumors, responding tumors, and
nonresponding tumors.
RNA Preparation.
RNA from rat tumors was prepared by using RNA STAT-60 (TEL-TEST
"B", INC., Friendswood, TX). Poly(A)+ RNA was
prepared by using Oligotex, mRNA mini-kit (QIAGEN Inc., Santa
Clarita, CA).
Probes Used in RNase Protection Assays and Northern Blots.
For RNase protection probes, rat aP2 (GenBank accession no.
U75581) and adipsin (GenBank accession no. M92059) were
amplified by RT-PCR. Amplified fragments were cloned into pGEM4Z and
confirmed by sequencing. Specificity of the probes for rat sequences
was verified by testing them against rat lung and brown fat RNA, known
to contain both aP2 and adipsin transcripts. A
cRNA probe was synthesized by linearizing the aP2 and adipsin
constructs with BamHI and EcoRI. respectively.
followed by in vitro transcription using T7 polymerase. For
Northern blots, human LPL and mouse PPAR cDNA
probes were kindly provided by Dr. Johan Auwerx (U 325 INSERM,
Institut Pasteur, F-59019 Lille, France) and Dr. Ronald M. Evans (Gene
Expression Laboratory, The Salk Institute for Biological
Studies, La Jolla California), respectively. Mouse SCD1 was
amplified by PCR (provided by Dr. Deepak Lala, Ligand Pharmaceuticals,
Inc. San Diego, CA; Ref. 17
) Human FAS cDNA
probe was provided by Dr. Marco Gottardis (Endocrine Oncology,
Bristol-Myers Squibb Pharmaceutical Research Institute, Princeton, NJ).
RNase Protection Assay.
RNase protection analysis was performed as described previously
(19)
. To control for RNA loading, a GAPDH probe was
included in all of the samples.
Northern Blot Analysis.
Poly(A)+ RNA (500 ng) was subjected to gel blot
analysis by standard methods. Blots were hybridized with various probes
labeled with [32P]dCTP using Ready To Go kit
(Pharmacia Biotech, Uppsala, Sweden) according to the method of Church
and Gilbert (20)
. Radioactive signals were quantified
using a PhosphorImager (Molecular Dynamics) and normalized against
GAPDH.
Quantitative Real-Time PCR.
Quantitative analysis by Real-time PCR was performed according to the
protocol of Perkin-Elmer. Total RNA (200 ng) in a final volume of 50
µl was transcribed into cDNA using murine leukemia virus
reverse transcriptase and random hexamer primers according to the
manufacturer’s protocol (Perkin-Elmer, Norwalk, CT). An additional
reaction not containing the enzyme serves as minus-RT control. After
synthesis, cDNAs were diluted with water to 200 µl and frozen in
10-µl aliquots. Real-time PCR was performed using gene-specific
primers and specific fluorescently labeled probes. Initial gel analysis
of the final amplification reaction confirmed that the fluorescent
signal resulted from the correct amplification product. In short, a
10-µl aliquot (10-ng RNA equivalents) of each cDNA was used for
Real-time PCR in a final volume of 50 µl, containing 300
nM of each forward and reverse primer and 100
nM of the fluorescent probe (according to the protocol
supplied by Perkin-Elmer). Together with the samples and minus-RT
controls, a serially diluted cDNA standard was analyzed in parallel.
Reactions were performed in an ABI PRISM 7700 Real-time PCR machine
(Perkin-Elmer) for 40 cycles of 15 s each at 95°C and of 60 s at 60°C. Quantitative analysis was performed using the threshold
procedure (Perkin-Elmer protocol), and relative amounts were calculated
from the standard curve. For all of the target genes analyzed, the
corresponding minus-RT controls in all of the samples showed that the
contribution of contaminating DNA to the signal was negligible. All of
the target gene expression was normalized to the expression of the
housekeeping gene 36B4 (21)
.
Primers and Probes for Real-Time PCR.
The primers and probes used were as follows: Rat 36B4 (Ref.
21
; GenBank accession no. X15096), Forward
5'-agatgcagcagatccgcat-3', Reverse 5'-ggatggccttgcgca-3', and Probe
JOE-aggctgtggtgctgatgggcaagaac-TAMRA; Rat aP2 (GenBank
accession no. U75581), Forward 5'-tccttcaaactgggtgtggaa-3', Reverse
5'-ccagggttatgatgctcttcact-3', and Probe
6FAM-tcctgtcatctggggtgatttcatcga-TAMRA; and Rat PPAR(Ref. 22
; GenBank accession no. AF156666), Forward
5'-ctgtgaagttcaatgcactggaat-3', Reverse 5'-atgggcttcacgttcagcaa-3', and
Probe 6FAM- cattattctcagtggagaccgcccagg-TAMRA.
Immunohistochemistry.
Affinity-purified rabbit antimurine ALBP/aP2 (dilution 1:2000; Ref.
23
) was a kind gift from Dr. David A Bernlohr
(Biochemistry Department, College of Biological Sciences, University of
Minnesota, St. Paul, MN). Tissues were fixed in zinc formalin (Anatech,
Battle Creek, MI) for 10 h, embedded in paraffin, and sectioned on
a sliding microtome at a thickness of 4 µm. Immunohistochemistry was
performed according to the standard protocol of PharMingen (San Diego,
CA). Positive staining was visualized by incubating the slides in
diaminobenzadine.
NMU417 Cells.
The NMU417 cell line was isolated from an explanted NMU-induced rat
mammary carcinoma at Ligand Pharmaceuticals by trypsin digestion and
was grown in DMEM with 10% FBS. The explanted rat mammary tumor cells
were cloned by plating in soft agar. Consistent with their malignant
phenotype and their ability to grow in soft agar, NMU417 cells
generated tumors when inoculated into nude mice (not shown).
Thymidine Incorporation Assay.
The method used for the determination of the incorporation of
radiolabeled thymidine was adopted from the procedure described by
Shrivastav and Paulson (24)
. NMU417 cells were plated in
96-well plates at a density of 500 cells/well and were incubated with
vehicle or LGD1069 for 4 days at 37°C. Subsequently, 1 µCi of
[5-3H]thymidine (43 Ci/mmol; Amersham Pharmacia
Biotech Inc., Piscataway, NJ) in culture medium was added to each well,
and the cells were incubated for an additional 6 h. Cells were
lysed, and incorporated [5-3H]thymidine was
assayed according to the scintillation proximity assay protocol
(Amersham Pharmacia Biotech Inc., Piscataway, NJ). Values represent the
mean cpm of incorporated thymidine from triplicate wells.
Anchorage-dependent Clonogenic Assay.
Clonogenic assays were performed as described previously
(25)
. Briefly, NMU417 cells were plated (500 cells/100-mm
dish) in triplicate and incubated with or without compound for 2 weeks.
Colonies were stained with crystal violet and counted.
Gene Expression Analysis.
For expression analysis, NMU417 cells were plated in 24-well dishes at
a density of 10,000 cells/well. LGD1069 (1 µM) or vehicle
was added the following day, and cells were harvested in
RLT-lysis buffer (QIAGEN Inc.) between 6 and 80 h after
treatment. Total RNA was prepared using the RNeasy Mini-Kit (QIAGEN
Inc.).
Statistical Analysis.
Statistical significance of differences between two groups was
determined by conducting a Student’s t test. Ps
0.05 were considered to be significant.
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RESULTS |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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.
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and RAR and express
high levels of RXR with lower levels of RXRß. The expression of
RXR was not changed after LGD1069 treatment, and the levels were
similar in control, responding, and nonresponding tumors. However the
expression of RXR was significantly increased in the responding
tumors (not shown). Expression of RARß was undetectable (not shown).
LGD1069 Induces the Expression of Adipocyte-related Genes in
Responding Tumors.
To evaluate whether treatment of NMU-induced mammary carcinomas with
LGD1069 caused an increase of adipocyte differentiation in responding
tumors, the expression levels of the adipocyte-related genes
aP2, adipsin, and PPAR were
determined in control, responding, and nonresponding tumors. Low levels
of aP2, adipsin and PPAR gene
expression were observed in both vehicle-treated control and
nonresponding tumors (Fig. 2,A and B)
. A significant increase of 8-fold
(P < 0.0003), 2.8-fold
(P < 0.0003), and 2.45-fold
(P < 0.008) in the expression level of
aP2, adipsin, and PPAR,
respectively, was observed in the tumors that responded to LGD1069
therapy when compared with control tumors (Fig. , 2A
and 2B)
. Also, a
significant increase of 5.8-fold (P < 0.0004), 3.7-fold (P < 0.0004), and 4.2-fold
(P < 0.0004) in the expression level of
aP2, adipsin, and PPAR genes,
respectively, was observed in responding tumors when compared with
nonresponding tumors.
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. Also, a significant increase of 3-fold
(P < 0.001) in LPL gene
expression was observed in responding tumors when compared with
nonresponding tumors. But the difference between the expression level
of FAS in responding and nonresponding tumors was not
significant (Fig. 2B)
. In contrast to the adipocyte-specific
genes, the expression levels of LPL and FAS were
significantly (P < 0.04) increased by 1.7-
and 1.9-fold, respectively, in nonresponding tumors when compared with
control tumors (Fig. 2B)
. No significant difference was
observed in the expression level of SCD1 either in the
responding or nonresponding tumors when compared with the control
tumors or between responding and nonresponding tumors.
In a separate experiment, naïve tumor-bearing animals were
given a single oral dose (100 mg/kg) of LGD1069 6 h prior to
harvesting the tumors for gene expression analyses. When compared with
vehicle-treated control tumors, higher aP2 expression levels
were observed in 75% of LGD1069-treated tumors (Fig. 2C)
.
The average aP2 expression level in tumors from
LGD1069-treated animals was 2.4-fold higher than the level in
vehicle-treated control tumors.
Histology and aP2 Protein Expression in Mammary Carcinomas.
The histology of LGD1069-responding tumors showed an increase in
stromal and surrounding tissue invasion as compared with
vehicle-treated control tumors. Responding tumors also had a decreased
number of cell layers and decreased cellular pleomorphism as indicated
by a well organized, more differentiated phenotype than control tumors
(Fig. 3)
. Morphologically, histologically and pathologically nonresponding
tumors were similar to the vehicle-treated control tumors. In
comparison to the control tumors a marked decrease in proliferating
cellular nuclear antigen (PCNA) immunostaining was observed in the
responding tumors (not shown). These results were similar to our
previously published results for bromodeoxyuridine incorporation
(8)
. No difference was observed between control and
nonresponding tumors.
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. Intense aP2-immunostaining was observed in the
adipocytes present in the stroma of regressing tumors, but more
importantly, aP2-immunostaining was also observed within the tumor
cells themselves (Fig. 3)
. Similar to control tumors, negligible
immunostaining of aP2 was observed in the nonresponding tumors.
LGD1069 Caused a Decrease in Proliferation and an Increase in
Adipocyte-related Gene Expression in Cloned Tumor Cells in
Vitro.
To determine the effect of LGD1069 treatment on tumor cells, outside
the complex tumor environment, tumor cells were cloned from an
explanted rat mammary carcinoma by plating cells in soft agar.
Consistent with their malignant phenotype and their ability to grow in
soft agar, NMU417 cells produced tumors when inoculated s.c. in nude
mice (not shown). To determine whether the isolated tumor cells were
responsive to LGD1069 treatment, cell growth kinetics were evaluated in
the cloned tumor cells. The growth of NMU417 cells was evaluated in two
separate assays, i.e., thymidine incorporation and
anchorage-dependent clonogenic growth. Treatment with LGD1069 (1
µM) inhibited cellular proliferation of NMU417
cells by 77% (P < 0.0002; Fig. 4A
). In clonogenic growth assays, colonies containing NMU417
cells were grouped mainly into two categories: large colonies
containing 180–200 cells and small colonies containing 10–30 cells.
LGD1069 (10-7 M) inhibited the formation of large
colonies by 87.5% (P < 0.05) and small
colonies by 58.8% (P < 0.02; Fig. 4B
).
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was evaluated in NMU417 cells. The cells were
treated with LGD1069 (1 µM) or vehicle over a
period of 80 h and analyzed for aP2 and
PPAR expression levels. Both genes were dramatically
increased by LGD1069 over the treatment period, whereas expression
levels in vehicle-treated controls remained almost constant (Fig. 5)
.
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DISCUSSION |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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correlate with LGD1069 mediated regression of rat
mammary carcinomas. Importantly, aP2 protein was also highly expressed
in regressing tumors when compared with control tumors and was
localized in the tumor cells in addition to the adipocytes present in
the tumor. In addition, LGD1069 inhibited the growth of and induced the
expression of aP2 and PPAR mRNA within a cloned tumor cell line
(NMU417). Hence, LGD1069 seems to initiate a process of adipocyte
differentiation in tumor cells leading to tumor regression.
The concept of differentiation induction therapy was described in 1927
in a report from Cushing and Wolbach (26)
that during
palliative treatment of an advanced childhood neuroblastoma, the
metastases of this malignant tumor were found to have spontaneously
differentiated into benign ganglioneuromas. More recently, the process
of differentiation induction is a well-established paradigm for the
treatment of some malignant diseases. Differentiation induction therapy
with 1-ß-D-arabinofuranosylcytosine (27)
,
hexamethylene bisacetamide (28)
, or 5-azadeoxycytidine
(29)
has been shown to be effective in treating acute
myelogenous leukemia; IFN- treatment induced differentiation of
hairy cell leukemia (30
, 31)
. Of the differentiation
inducing cancer therapeutics tested, RA derivatives (retinoids) have
shown the most potential. Therapy with retinoids has demonstrated
substantial clinical benefit in t(15;17) positive acute promyelocytic
leukemia (32
, 33)
in juvenile chronic myelogenous leukemia
(34)
and T-cell lymphoma (35)
. Recently, we
have observed the induction of adipocyte differentiation in clinical
samples from liposarcoma patients after treatment with Targretin
(LGD1069).6
Similarly, the induction of adipocyte
differentiation in human liposarcoma after treatment with the
PPAR-ligand troglitazone has also been shown (13)
. In
the present study, we demonstrate the induction of adipocyte
differentiation in solid tumors of the mammary gland, or in
mammary carcinomas by LGD1069 therapy. The process of adipocyte
differentiation was characterized by the induction of
adipocyte-specific gene expression and the loss of growth that
correlated with the regression of carcinogen-induced rat mammary
carcinoma.
Earlier, we demonstrated that the RAR and RXR pan-agonist 9-cis-RA induced cell differentiation that led to terminal cell division followed by cell death in the human promyelocytic leukemia cell line HL-60 (36) . Similarly, in human breast cancer cell cultures, we have observed the induction of differentiation by lipid accumulation and inhibition of proliferation by the RXR ligand, LGD1069.5 In the NMU-induced rat mammary tumor model, we have also shown that LGD1069 inhibited the proliferation of tumor cells in vivo (8) . Now, in the present study, we observed the expression of the adipocyte-specific protein aP2 in the tumor cells of regressing tumors during LGD1069 treatment. Additionally, we demonstrated the inhibition of cell growth and induction of adipocyte-related gene expression in tumor cells (NMU417) isolated and cloned from an explanted NMU-induced rat mammary tumor. Because we did not observe any morphological changes consistent with the aP2-expressing tumor cells differentiating into adipocytes, we are not implying that the tumor cells are undergoing transdifferentiation into mature adipocytes. We hypothesize that LGD1069 induces adipocyte differentiation within the tumor cells of NMU-induced mammary carcinomas that leads to terminal cell division followed by cell death.
The induction by LGD1069 of aP2 mRNA expression in rat mammary tumors occurs rapidly, in that when naïve tumor-bearing animals were given a single oral dose of LGD1069, they showed increased aP2 expression in the tumors in as little as 6 h after administration. Interestingly, increased aP2expression was not seen in all of the naïve tumors exposed to LGD1069 but only in 75% of them. These numbers correlated well with our previous observation that LGD1069 causes complete regression in 72% of NMU-induced rat mammary carcinoma (8) . This implies that the induction of aP2expression may be an early predictive marker of responsiveness to LGD1069 therapy and may allow one to subset tumors into responders versus nonresponders based on the ability of LGD1069 to induce adipocyte-related gene expression.
The RXRs function as homodimers or as heterodimers with various nuclear
receptors such as the PPARs, liver X receptors, farnesoid X
receptors, RARs, vitamin D receptors, and thyroid hormone receptors
(37)
. Because RXR ligands induced lipid accumulation and
the expression of FAS gene in human breast cancer cell
lines,5
and LGD1069 induced adipocyte-related gene
expression in responding rat mammary tumors, it is possible that
nuclear receptors involved in lipid homeostasis are the heterodimeric
partners of RXR involved in the regression of NMU-induced tumors.
RXR/PPAR is a permissive heterodimer because ligands of either
receptor activate this heterodimer in a variety of cells including
preadipocytes (11)
. LGD1069 treatment induced PPAR
expression in NMU-induced mammary tumors responding to LGD1069. Thus,
one possible heterodimer important in this model may be the RXR/PPAR
heterodimer, which is known to transcriptionally induce aP2 and PPAR
resulting in adipocyte differentiation (38)
. We and others
(11
, 12)
have also shown that the activity of RXR/PPAR
is synergistically increased in preadipocytes when a RXR-ligand is
combined with a PPAR ligand. This synergistic activity may have
implications for the treatment of mammary carcinoma. It will be
interesting to examine the therapeutic effects of combining a RXR
ligand with a PPAR ligand in the NMU-induced rat mammary carcinoma
model. Even when PPAR expression in vehicle-treated control
tumors is low, a single dose of LGD1069 induces aP2 expression;
therefore, the RXR/PPAR heterodimer may not be the only heterodimer
important for the action of LGD1069 in the NMU-carcinogenesis model.
Other heterodimeric partners, e.g., liver X receptors, which
are known to be involved in lipid homeostasis, may also play a role in
LGD1069 action in NMU-induced rat tumors.
From a therapeutic point of view, it was important to determine how to initiate or trigger the process of LGD1069-mediated regression in mammary tumors. This information may lead to the discovery of additional cancer therapeutic targets for RXR ligands. In the present report, we demonstrate that Targretin (LGD1069) induced adipocyte-specific gene expression in responding mammary carcinomas and that increased gene expression was correlated with the regression of these tumors. LGD1069 also inhibited the proliferation of NMU-induced cloned tumor cells and induced adipocyte-related gene expression in cloned NMU-induced mammary tumor cells in vitro. Thus, it appears that in the NMU-carcinogenesis model, RXR ligands cause tumor regression by inducing adipocyte differentiation in the tumor cells, which is followed by terminal cell division and cell death.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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1 Present address: Bayer Corporation, West Haven,
CT 06516. 
2 Present address: X-Ceptor Therapeutics, San
Diego, CA 92121.
3 To whom requests for reprints should be
addressed, at Department of Nuclear Receptor Discovery, Ligand
Pharmaceuticals Inc., 10275 Science Center Drive, San Diego, CA 92121.
Phone: (858) 550-7500; Fax: (858) 550-7730;
4 The abbreviations used are: TAM,
tamoxifen; NMU, N-nitroso-N-methylurea;
RXR, retinoid X receptor; LPL, lipoprotein lipase; aP2, adipocyte P2;
PPAR, peroxisome proliferator-activated receptor ; SCD1,
stearoyl-CoA desaturase-1; FAS, fatty acid synthase; RT, reverse
transcription; GAPDH, glyceraldehyde 3-phosphate dehydrogenase; RA,
retinoic acid; RAR, RA receptor.
5 V. R. Agarwal, T. Hermann, R. P. Bissonnette, W.
W. Lamph. The RXR-agonist LGD1069 (Targretin) inhibits cell
proliferation and causes lipid accumulation in both PPAR-positive
and PPAR-negative human breast cancer cells, manuscript in
preparation.
6 P. J. A. Davies and W. W. Lamph,
unpublished observations.
Received 3/23/00. Accepted 9/ 1/00.
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,
statistical significance (P < 0.02) between
control and responding tumors; 
, statistical significance
(P < 0.001) between responding and
nonresponding tumors; +, statistical significance
(P < 0.04) between nonresponding and control
tumors. In C, LGD1069 induces the expression in
naïve tumor-bearing animals of aP2 in as few as
6 h after p.o. administration. Expression was examined by
Real-time PCR. The expression of the housekeeping gene 36B4
was used to normalize gene expression levels. The data shown are
normalized to the average expression level (arbitrary units) in
vehicle-treated control tumors and are the average ± SD
of duplicate determinations from each sample.
