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,
1
* The Albert Einstein Cancer Center,
Department of Developmental and Molecular Biology,
Department of Medicine and
§ Department of Biochemistry, Albert Einstein College of Medicine, Bronx, New York 10461, USA;
¶ Smith College Lyman Conservatory, Northampton, Massachusetts 01636, USA;
|| Dana Farber Cancer Institute, Boston, Massachusetts 02115, USA
1Correspondence: The Albert Einstein Cancer Center, Departments of Medicine, and Developmental and Molecular Biology, Albert Einstein College of Medicine, Chanin 302, 1300 Morris Park Ave, Bronx, New York 10461, USA. E-mail pestell{at}aecom.yu.edu
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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and a chimeric
ecdysone receptor under control of a modified MMTV-LTR, which targets
mammary gland expression, were used. These transgenic ‘receptor’
lines were crossed with transgenic ‘enhancer’ lines in which the
ecdysone/RXR binding site induced ligand-dependent expression of
transgenic ß-galactosidase. Pharmacokinetic analysis of a highly
bioactive ligand (ponasterone A), identified through screening
ecdysteroids from local plants, demonstrated sustained release and
transgene expression in vivo. This transgenic model with
both tightly regulated and homogeneous transgene expression, which was
sustained in vivo using ligands readily extracted from
local flora, has broad practical applicability for genetic analysis of
mammary gland disease.—Albanese, C., Reutens, A. T., Bouzahzah,
B., Fu, M., D’amico, M., Link, T., Nicholson, R., Depinho, R. A.,
Pestell, R. G. Sustained mammary gland-directed, ponasterone
A-inducible expression in transgenic mice.
Key Words: ecdysteroids • gland-specific transgenics • DNA binding site
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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.
Binary systems, in which one mouse line contains an activator of
expression and the second mouse line contains the silenced gene of
interest, have been developed to examine the expression of potentially
lethal or hypomorphic genes. Furthermore, inasmuch as the acquisition
of somatic mutations in the adult contribute to the onset and
progression of tumorigenesis, genetic manipulation postnatally in a
defined tissue is likely to recapitulate oncogenic mechanisms more
faithfully and thus provide a more informative model for the study of
disease.
Transgenic approaches to controlled inducible misexpression of target
genes have used transactivators such as the VP16 and Gal4/UAS
activation system driven by tissue-specific promoters (2
, 3)
. The tetracycline-regulated system was developed to provide
temporal and spatial control of gene expression (4
, 5)
. In
the presence of the ligand, the transactivator tTA is bound to the tetO
promoter, and gene activity is silent until the ligand is removed
(4
, 5)
. Alternatively, in the reverse tTA system,
transcriptional activity is induced by the addition of ligand.
Successful application of this system to transgenics (6)
has been balanced by reports of mosaic induction, background leakiness,
and poor expression of the transactivator, perhaps related in part to
the inefficient processing of the tetR gene in mammalian
cells, which may limit the utility of this approach for the study of
cancer (1)
. Steroid hormone inducible systems have
centered on the expression of chimeric receptors that bind nontoxic
hormone ligands. Estrogen receptor chimeric transgenics induced by
tamoxifen show promise (7)
, but evidence of mosaic
transgene expression in target tissues (8)
and the need to
use toxic and teratogenic doses for robust expression (9)
suggest that components of this system require further modifications.
The ligand for the ‘ecdysone’ system is a molting hormone, 20
OH-ecdysone, which normally binds a heterodimer of the
Drosophila ecdysone receptor (EcR) and the product of the
ultraspiracle gene in the context of a specific DNA binding
site (10)
(11)
. Ecdysteroids are neither
teratogenic nor known to affect mammalian physiology. A chimeric
receptor consisting of the VP16 transactivation domain fused to an
amino-terminal truncation of the modified EcR (VgEcR) was shown to
transactivate a reporter gene consisting of multimeric ecdysone
enhancer binding sites with higher inducibility than comparable
tet-regulated reporters (12)
. Furthermore, the basal level
activity of the enhancer was low in mammalian cells, consistent with
the potential utility of this system for transgenesis
(12)
. Despite these promising features, sustained targeted
transgene expression has not been reported, perhaps related to the lack
of published pharmacokinetic data, tissue distribution analysis, and
the unavailability of inexpensive ligands (1)
. In the
current studies we describe the development and successful
implementation of a system for targeted mammary gland-specific
expression in transgenic mice, the purification of ponasterone A, a
potent ecdysteroid ligand, from plants, and the use of sustained
release ponasterone A.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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MTVLuc (12)
linked to
the minimal TK promoter in the pA3LUC reporter
plasmid (13)
. The modified MMTV promoter from MMTVp206
(14
, 15)
that incorporates the 5'UTR of v-Ha-ras was
inserted into a vector containing a modified ecdysone receptor VgEcR (a
gift from Dr. R. Evans) to form MMTV-VgEcR. The ß-galactosidase cDNA,
a derivative of pPD16.51, with a eukaryotic polyadenylation site
(16)
(a gift from Dr. A. Fire), was inserted into the EcR
target plasmid (E/GRE)3MTV (from Dr. R.
Evans). DNA constructions were prepared and injected into CBA/C57Bl6
fertilized eggs. The MMTV-VgEcR and CMV-RXR constructions were
coinjected. Genotyping was performed by genomic Southern blot analysis.
Transgenic lines were bred into the FVB genetic strain of mice for at
least four generations. The reporter line
(E/GRE)3ß-Gal was bred to the receptor line and
the presence of the three transgenes was confirmed by genomic Southern
analysis. In addition, a distinct plasmid, pVgRXR, was obtained from
InVitrogen (Carlsbad, Calif.) and used in cell culture experiments as
indicated in the text.
Cell culture, DNA transfection, and luciferase assays
Cell culture, DNA transfection, and luciferase assays were
performed as described previously (13)
. 293T (BOSC) cells
(from Dr. D. Baltimore) were maintained in Dulbecco’s modified Eagles
medium with 10% fetal calf serum and 1% penicillin/streptomycin.
Cells were transfected by calcium phosphate precipitation with
pVgEcR/RXR and (E/GRE)6TKLUC. Six hours after
addition of the precipitate, the media were changed and treatment was
performed for the time points indicated. At least two different plasmid
preparations of each construct were used. Luciferase assays were
performed at room temperature using an Autolumat LB 953 (EG&G
Berthold). Luciferase content was measured by calculating the light
emitted during the initial 10 s of the reaction and the values are
expressed in arbitrary light units. Statistical analysis was performed
using the Mann Whitney U test.
Ponasterone purification
The leaves of candidate species from the Podocarpus
or Taxus genus of evergreen plants were ground in liquid
nitrogen, refluxed in methanol, and subjected to high-pressure liquid
chromatography (HPLC) over a micro-Bondpak C-18 column (3.9x300 mm
column, Waters Inc., Milford, Mass.). Fractions (20 ml) of a 15–50%
acetonitrile gradient were collected and lyophilized. The dried samples
were resuspended in 100% ethanol and stored at -20°C. Ponasterone A
was extracted from mammary tissue from ponasterone A-treated mice by
grinding the mammary tissue in liquid nitrogen, refluxing overnight in
ethanol and centrifuging at 5000 g for 20 min. The
supernatant was dried and subjected to HPLC as stated above
(17)
. In addition, purified ponasterone was obtained from
Dr. K. Nakanishi and was used as indicated. Delayed release ponasterone
A and placebo pellets were made by Innovative Research (Innovative
Research of America, Sarasota, Fla.).
Immunohistochemistry and ß-galactosidase assays
Mammary tissue was taken from lactating mice 1 day postpartum.
For ß-galactosidase staining, 5 mm cubes of tissue were fixed in 2%
paraformaldehyde/0.02% glutaraldehyde for 1 h, washed with
phosphate-buffered saline (PBS) twice, and stained at 30°C with 0.1%
4-chloro-5-bromo-3 indol-b-D-pyranoside in 2 mM
MgCl2, 5 mM EGTA, 0.02% (v/v) Nonidet P-40, 5 mM
K3Fe(CN)6, and 5 mM
K4Fe(CN)6.H20
for 28 h (18)
. The samples were embedded in paraffin,
counterstained with nuclear fast red, and examined for blue nuclei. For
immunohistochemistry, samples were frozen and sectioned on a cryostat.
Sections were fixed in cold acetone, treated with hydrogen peroxide,
and incubated in either anti-VP16 primary antibody (V-20; Santa Cruz
Biotechnology, Santa Cruz, Calif.) or anti-RXR antibody (D-20; Santa
Cruz) at a 1:2000 dilution overnight at 4°C. The samples were washed
with PBS and incubated with a biotinylated secondary antibody (Santa
Cruz) at a 1:1000 dilution for 30 min at room temperature. The sections
were incubated for 30 min at room temperature in avidin-horseradish
peroxidase and stained with diaminobenzadine.
Reagents
20 OH-ecdysone was a gift from Dr. Peter Cherbas. Inokosterone
and ponasterone A were gifts from Dr. Koji Nakanishi. Muristerone was
obtained from Sigma (St. Louis, Mo); chemicals were obtained from
Fisher (Fairlawn, N.J.) and were of HPLC grade.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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. In previous studies, a
chimeric ecdysone receptor, in which the amino-terminal truncation of a
modified EcR was attached to the VP16 activation domain (VgEcR), was
shown in reporter assays to enhance ecdysteroid-induced reporter
activity compared with the parental EcR receptor (12)
.
Comparison was therefore made of the relative activity of these
ecdysteroids in a synthetic luciferase reporter assay driven by a
multimeric optimized enhancer element for the modified chimeric
VgEcR/RXR receptor. Dose response curves were conducted with each
ligand on cells transfected with the reporter gene and an expression
plasmid for the bi-cistronic VgEcR/RXR receptor, and the relative
activity was compared with vehicle (Fig. 1A
). Analyses for basal and ligand-induced activity performed
after 24 h of treatment showed that ponasterone A is ~1000-fold
more active than 20 OH-ecdysone. Ponasterone A and muristerone
exhibited similar potencies (Fig. 1A
). Basal activity of the
reporter was inhibited by receptor in the absence of ligand by 40%
(data not shown) and no transcriptional activation was observed in the
absence of cotransfected RXR (data not shown).
|
To identify a source of bioactive ponasterone A, candidate flora were
screened (Fig. 1B
). Ponasterone A was extracted as described
previously (17)
. Activity of the extracted material was
compared with the activities of purified ponasterone A. Relative
abundance was expressed as milligrams of ponasterone A/kilogram of
material. Podocarpus macrophyllus D. Don and P.
macrophyllus v. Maki both produced ponasterone A as did
Taxus cuspidata, T. chinensis, T.
canadasis, and T. x media ‘Hicksii’ (Fig. 1B
).
ponasterone A delivery to the mammary gland
As ponasterone A was the most bioactive ecdysteroid in tissue
culture reporter assays, the in vivo serum and tissue
half-life of ponasterone A activity was determined in the mouse. To
improve sensitivity of the reporter system and accurately measure low
ponasterone A concentrations, a reporter
(E/GRE)6TKLUC was made that enhanced sensitivity
by fourfold (Fig. 2A
). Ponasterone A activity was assessed in serum and tissues
using this reporter. Serum samples from the mouse were assayed using
the reporter gene with comparison made to a standard curve performed
using purified ponasterone A (a gift from Dr. K. Nakanishi). Serum
levels of ponasterone A were readily detectable 15 min after
intraperitoneal (i.p.) injection (Fig. 2B
). At least four
different animals were compared at each time point. The half-life of
serum ponasterone A activity, determined through sequential analysis
after i.p. injection, demonstrated rapid clearance with an activity
half-life of 48 min as determined by plotting the serum concentration
vs. time on a semilogarithimic scale (20)
.
|
As the goal of the current studies was to direct mammary
gland-specific transgene expression, the concentration of ponasterone A
was determined in the mammary gland 24 h after i.p. injection of
ponasterone A. HPLC purification of ponasterone A within the mammary
gland was performed as described in Materials and Methods. The activity
of the HPLC-purified material was then assessed using the
(E/GRE)6TKLUC reporter assay with comparison made
to the standard curve. The ponasterone A concentrations were 400–500
pg/g of tissue (Fig. 2C
). Sustained delivery of steroids
through subcutaneous (s.c.) implantation has previously provided
bioactive levels for treatment of steroid deficiency states in mice and
humans (21)
. Serum ponasterone A activity was therefore
assessed after s.c. implantation of ponasterone A pellets. Ponasterone
A was incorporated into 21 day release implantation pellets and placed
under the skin of mice as described in Materials and Methods. Blood was
taken at days 4 and 15 postimplantation and assayed. Ponasterone A was
detected in mammary tissue (Fig. 2C
). In mammary gland
tissue from mice carrying the 100 µg implant, ponasterone A
concentrations were 3–4 ng/g of tissue. Ponasterone A activity was
undetectable in peripheral blood (data not shown).
Mammary gland-targeted VgEcR/RXR transgenic mice
Transgenic mice were generated in which the enhancer line
expressed an ecdysone-responsive ß-galactosidase reporter gene,
(E/GRE)3MTVß-Gal, as a marker of
ecdysteroid-regulated gene expression (Fig. 3A
). VgEcR was targeted to the mammary gland through the use
of a modified MMTV promoter construction (MMTVp206) (15
, 22)
, which enhances mammary gland-specific transgene expression
through the addition of the 5' UTR from v-Ha-ras (15)
.
Three founder lines were established. The receptor and enhancer mice
were bred to form triply transgenic reporter/ecdysone enhancer (TR/EE)
lines and the integrity of the triply transgenic mice (TR/EE lines) was
confirmed through genomic Southern analysis (Fig. 3B, C
).
The presence of the RXR transgene in the TR/EE lines was confirmed
by genomic Southern blot analysis (Fig. 3B
, lane 2). The
presence of the RXR protein in the mammary gland tissue of the TR/EE
lines was confirmed by Western blot analysis with a comparison made to
the immunoreactivity of in vitro translated RXR (right
panel, Fig. 3B
). The presence of RXR in the mammary gland
of the TR/EE lines was confirmed by immunohistochemistry using an
RXR-specific antibody as described in Materials and Methods (lower
left panel, Fig. 3B
). Minimal background staining of the
tissue was observed in the absence of primary antibody (lower right
panel, Fig. 3B
).
|
The presence of the VgEcR transgene in the TR/EE lines was confirmed by
genomic Southern analysis (Fig. 3C
). The presence of the
VgEcR receptor expression was assessed in the mammary gland tissue from
lactating TR/EE lines using immunohistochemistry and a specific
antibody to VP16 (Fig. 3C
). Immunopositivity was observed
throughout the mammary epithelium (depicted as EC in the left panel of
Fig. 3C
). No staining was observed within adjacent blood
vessels (blood vessel (depicted as BV in the left panel of Fig. 3C
). The mammary glands of nontransgenic control mice were
immunonegative for the VgEcR (Fig. 3C
, right panel). mRNA
for the VgEcR was detectable by Northern blot analysis in transgenic
mammary gland but was not detectable in heart, liver, or salivary gland
(data not shown).
To evaluate the tissue-specific expression and functional activity of
the triply transgenic mice, the TR/EE mice were treated with
ponasterone A and tissues were analyzed for ß-galactosidase abundance
after 24 h. Costaining with fast red was performed to identify
nuclei. The basal level ß-galactosidase abundance was undetectable in
the mammary gland in the absence of ponasterone A (Fig. 4A
). Ponasterone A (750 µg) induced ß-galactosidase
activity uniformly throughout the mammary gland (Fig. 4B
).
Analyses performed of other tissues showed basal low but detectable
levels of ß-galactosidase activity in the salivary gland (Fig. 4C
), but no activity in the liver, heart, spleen (Fig. 4D-F
), kidney, ureter, ovary, or uterus (data not
shown). The low level of expression within the salivary gland is
consistent with previous reports that the MMTV promoter may direct
expression to the salivary gland.
|
In many transgenic experiments, sustained expression may be required.
Ponasterone A pellets were implanted s.c. in the subscapular region of
the enhancer/receptor mice using a trocar according to the
manufacturer’s direction. ß-Galactosidase staining of the mammary
gland tissue performed after 10 days (Fig. 5
) was observed to be uniform throughout the mammary epithelium. These
studies demonstrate that sustained transgene expression can be
maintained through ponasterone A implantation.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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24
25)
. Alternatively, chimeric
transcription factors or mutant receptors can be regulated by
tetracycline, mifepristone, tamoxifen, or synthetic small molecules
(4
, 6
, 26)
. Transgenic systems for functional analysis of
genes involved in mammary gland development and disease, particularly
tumorigenesis, should ideally combine features of low basal level
activity, robust induction by a ligand that is rapidly cleared from the
serum, and have homogeneous transgene expression in mammary epithelium.
The current studies describe the generation of a transgenic mouse model
in which spatial- and temporal-specific mammary gland transgene
expression regulated by the ecdysteroid ponasterone A combines these
features.
A practical limitation in the past for these studies was the ready
availability of an ecdysteroid ligand and evidence of its sustained
function in vivo. In the current studies, serum clearance of
ponasterone A was rapid with an activity half-life of 48 min. We
identified a source of bioactive ponasterone A and extracted milligram
quantities using simple techniques (17
and data not shown)
and demonstrated sustained transgene expression induced by s.c.
implants. Transgene expression was induced within 24 h and
sustained using the s.c. implantation of ponasterone A pellets.
Bioactive ponasterone A was readily measured in the mammary tissue of
the transgenic mice implanted with ponasterone A pellets. The triply
transgenic mice have both a similar litter size to control litter mates
(Tg. 8.0±1.2, n=14 litters vs. Ctrl 9.0±1.5,
n=15 litters) and normal breast morphology by mammary gland
whole mount (not shown). Other steroid-inducible transgenic systems
have also shown promising results. The steroid ligand tamoxifen induced
partial reporter gene expression within 24 h in transgenic mice,
with evidence of activity sustained for 4 days (8)
. The
intracellular level of tamoxifen appeared to be a limiting factor in
the kinetics of inducing transgene expression (8)
. In
cultured cells (27)
and in transgenic mice
(28)
, chimeric progesterone receptors were modulated by
the anti-progesterone RU486. Although concerns exist that the
concentrations of RU486 required to regulate the chimera in transgenic
mice antagonize endogenous progesterone and glucocorticoid receptors,
the short half-life of RU486 suggests that the side effects would be
transient, implying promise for this system’s applicability to
transgenics. The steroid ligands RU486 and tamoxifen can affect
endogenous mammary gland nuclear receptors, breast epithelial cell
cycle progression, and cellular proliferation (29)
,
limiting their potential applicability for analysis of mammary gland
gene function. In contrast, ponasterone A did not affect the cell cycle
in MCF7 cells at concentrations that induced reporter activity by
50-fold (data not shown), implying that ponasterone A may be an ideal
ligand for the analysis of genes involved in cell cycle regulation.
In the current studies, transgene expression within the mammary
epithelium of the receptor/enhancer transgenic mice was homogeneous.
Alternative mammary gland-directed promoters, including the whey
acidic protein gene, resulted in mosaic transgene expression
(30)
; its activity during puberty and pregnancy cannot be
controlled experimentally (31)
. Our findings of
homogeneous transgene expression contrast with results using a
different MMTV-LTR in which mosaic expression and heterogeneous
staining of ß-galactosidase presented practical limitations for
analysis of mammary gland-specific gene function (18)
. The
predominant expression of the inducible transgene in mammary tissue we
observed also contrasts with previous studies in which ectopic
expression of the tet-regulated expression system was observed
(18)
. We did not observe ectopic transgene expression in
the heart, lung, or spleen after the addition of ponasterone A. In
contrast, ectopic basal and tet-induced transgene expression was
observed in these same tissues of MMTV-LTR-driven, tet-regulated
transgenic mice (18)
. It is likely that the improved
expression profile and tissue specificity we observed was due to the
type of MMTV promoter (14)
driving the expression of the
VgEcR.
The enhancer/receptor transgenic mice described here have broad
applicability for genetic analysis of mammary gland development and
disease states. The low basal level and rapid induction of transgene
expression observed with the ecdysone system may be advantageous in
regulating CRE-mediated expression and somatic excision. The low basal
level expression of transgenic mice expressing tamoxifen-regulated CRE
expression was associated with successful ligand-inducible excision of
a floxed CAT cassette targeted to the skin (8)
as
well as CRE-mediated recombination targeted to the neural tube during
development (9)
. As the ecdysone system has been used to
induce FLP recombinase in cultured cells (32)
, the current
studies suggest that ponasterone A-induced, mammary gland-specific CRE
expression could be used for mammary gland genetic analysis in
vivo. This transgenic model may be used to analyze the function of
putative mammary gland tumor suppressors when mated with mammary gland
oncogenic mouse models, for analysis of functional interaction between
cooperating oncogenes, and for analysis of dominantly acting oncogenes
induced postnatally (33
, 34)
.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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REFERENCES |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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