Abstract

1
Standardization and validation of Adenoviral
transduction of an AR positive cell line with an
MMTV-Luc reporter for endocrine screening P.
Hartig, K . Bobseine, M. Cardon, C. Lambright
and L. E. Gray, Jr. USEPA, ORD, NHEERL,
Reproductive Toxicology Division, RTP, NC
27711
Abstract The discovery of xenobiotics which
interfere with androgen activity has highlighted
the need to assess chemicals for their ability to
modulate DHT-receptor binding. Previous test
systems have used cells transfected with plasmid
containing a reporter gene. Here we report the
novel use of transduction for gene delivery and
assessment of the modulation of DHT-induced gene
activation. Transduction, the ability of
replication-defective viruses to deliver
biologically competent genes, is a well
understood biological process which has been
utilized to repair defective genes in humans as
well as to express exogenous genes in rodent
models. Human breast carcinoma cells
(MDA-MB-453) containing endogenous copies of the
AR and GR were transduced with
replication-defective human adenovirus type 5
containing the luciferase reporter gene driven by
the AR GR responsive glucocorticoid-inducible
hormone response element found with the mammary
tumor virus LTR (Ad/MLUC7). Cells were
subcultured in 96 well plates, transduced with
virus, exposed to chemicals, incubated 48 hrs,
lysed and assayed for luciferase. Luc gene
expression was induced in a dose-dependent manner
by DHT, E2, Dex and high concentrations of OHF,
and M2, and inhibited by AR antagonist OHF,
OH-DDE, HPTE and M1. E2-induced expression was
blocked with OHF, but not the anti-estrogen ICI.
Transduction of MDA-MB-453 cells with MMTV-Luc
are nearly identical to those obtained with cell
lines stably expressing MMTV-Luc. In summary,
this assay positively identified all chemicals
examined and induction was found to be sensitive
and displayed a high fold (approx 100 X)
induction. Transduction can be rapidly applied to
other cell lines and utilized to deliver
receptors and reporter genes quickly (This does
not reflect EPA policy).
DHT-Induced Luciferase Expression in MDA-453
Cells Transduced with
OH-DDE Inhibits DHT-induced Luciferase Expression
in Adenovirus
Adenovirus
Transduced MDA-453 Cells
60
25.0
Results DHT-induced luciferase expression in
MDA-453 cells transduced with the androgen and
glucocorticoid regulated luciferase
gene. Combinations of Dexamethasone and DHT or
Medroxyprogesterone Acetate (MPA) and DHT
induced luciferase expression in transduced
MDA-453 cells which is less than additive. HPTE,
OH-DDE, M2 and M1 all inhibit DHT-induced
luciferase expression in MDA-453 cells transduced
with the androgen and glucocorticoid regulated
luciferase gene. M2 but not M1 induced
luciferase expression in MDA-453 cells
transduced with the androgen and glucocorticoid
regulated luciferase gene. E2-induced luciferase
expression in transduced MDA-453 cells is
blocked to a greater extent with an anti-androgen
(OHF) than with an anti-estrogen (ICI) .
No OHF
50
OH-DDE
1uM OHF DHT
OH-DDE 0.1nM DHT
20.0
40
30
15.0
Relative Light Units
Relative Light Units
20
10.0
10
5.0
0
ET-OH
1 fM
10 fM
100 fM
1 pM
10 pM
100 pM
1 nM
10 nM
100 nM
1 uM
10 uM
0.5
5.0
1.9
2.4
0.6
0.6
2.1
16.6
41.0
47.1
39.6
33.5
No OHF
0.5
0.3
0.4
0.3
0.4
0.4
0.6
4.1
15.7
28.9
28.8
1uM OHF
0.0
DHT Concentration
0.1 nM
0.05 uM
0.2 uM
0.5 uM
1.0 uM
10 uM
0.1 nM
ET-OH
DHT 1
Introduction The Safe Drinking Water Act and
the Food Quality Protection Act require the EPA
develop a high-throughput screening program to
identify potential endocrine disrupting
chemicals. A prudent screening program should
include both in vivo and in vitro assays.
Therefore, the feasibility of several in vitro
screening concepts was assessed. The initial
assessment was of hAR binding and/or
transcriptional activation protocols.
Historically this type of in vitro assay involves
the transfection of a tester cell line with a
plasmid base receptor and the reporter followed
by chemical exposure and measurement of the
modulation of gene expression. These types of
assays require relatively large numbers of cells,
costly transfection material and each
transfection event introduces yet another source
of experimental variation. One approach to
reduce this variation is to generate stable cell
lines which contain the genes of interest (see
posters 346 351). Another approach is to
deliver the genes via replication-defective
Adenovirus (transduction). Viral transduction is
a precise and reproducible way of delivering
genes in a cost-effective manner. In this study
we have assessed the ability of an Adenovirus to
transduce the human breast cancer line MDA-MB-453
(AR/GR/PR-/ERa- /weak ERb) with a luciferase
gene regulated by the glucocorticoid-inducible
hormone response element found in the mouse
mammary tumor virus (MMTV) LTR. Utilization of
MDA cells, which already contained an endogenous
AR, simplified the development of this assay by
reducing the transduction requirements to a
single reporter gene.
OH-DDE
OH-DDE
OH-DDE
OH-DDE
OH-DDE
DHT
uM OHF
3.1
3.2
1.9
2.0
1.7
3.9
20.1
1.5
OH-DDE
Effect of ICI and OHF on E2-induced Luciferase
Expression in Adenovirus
10.0
3.0
2.2
1.9
5.3
OH-DDE 0.1nM DHT
Transduced MDA-453 Cells
100.0
E2
E2 1uM ICI
E2 1uM OHF
Conclusions Adenovirus transduction provides a
valuable method for delivering exogenous genes.
The behavior of the transduced genes can be
utilized to assess endocrine disrupting
chemicals. The androgen and glucocorticoid
regulated luciferase gene (MMTV promotor)
responds similarly to chemical stimulus whether
it is delivered by transfection, transduction or
stably integrated into the cellular
genome. Transduction utilizes the innate
cellular entry mechanisms of the parental virus.
Because adenovirus can enter a wide variety of
cells, this method should allow the efficient and
cost effective delivery of genes to various cell
lines with different compliments of endogenous
receptors. The ability to easily transduce
numerous cell lines should facilitate the studies
of chemical/receptor interaction.
10.0
Effect of M1 M2 on DHT-induced Luciferase
Expression in Adenovirus
Relative Light Units
Transduced MDA-453 Cells
Materials and Methods Ad/MLUC7, which contains
the luciferase gene regulated by the
glucocorticoid-inducible hormone response element
found in the mammary tumor virus (MTV) LTR, was a
gift from Cary Weinberger (NIEHS).The virus had a
necessary early gene (Adenoviral gene E1)
replaced with the reporter gene.The virus is
replication deficient and can only be propagated
in a complementary cell line, such as the
transformed human embryonal kidney cell line 293,
which contains a copy of the E1 gene. Low
passage level 293 cells were purchased from
Microbix Biosystems Inc. (Ontario). The human
breast carcinoma cell line MDA-MB-453 (MDA) was
obtained from American Type Culture Collection,
cat ATCC HTB 131 ( Rockville, MD ). Serum was
purchased from Hyclone (Logan, UT ). All other
tissue culture reagents were purchased from GIBCO
BRL (Grand Island NY). Monolayer 293 cell
cultures were maintained at 370C, 100 humidity
in 5 CO2 in growth medium MEM (Cat11435-039)
supplemented with amphotericin B ( 2.5 µg /ml),
penicillin and streptomycin( 100U and 100µg/ml
respectively), glucose (3.5 mg/ml ), glutamine
(300µg/ml ), Hepes pH 7.3 (2.4 mg/ml), NaHCO3 (
2.2 mg/ml) and 10 heat inactivated (56oC 30
min.) Fetal Bovine Serum (FBS). Cells were
suspended with citric saline (KCL10mg/ml,
Na3C6H5O7 2H2O 4.4mg/ml) and subcultured 13
when 80 confluent. MDA cells were maintained at
370C, 100 humidity with no supplemental CO2 in
Leibovitzs L-15 medium supplemented with 100 U
penicillin, 100 µg/ml streptomycin and 0.25 µg/ml
amphotericin B and 10 FBS ( not heat
inactivated). MDA cells were suspended by
standard trypsinization techniques and
subcultured 1 4 weekly. Initial viral stocks
contained about 1x 109 plaque-forming units
(pfu)/ml. Virus was propagated by inoculating
150 mM dishes containing 293 cells which were 80
confluent. Medium was removed, inocula
instilled, dishes rocked every 15 min for 1 hr.
Then 20 mls fresh media were added. The cells
were incubated 2-3 days until 95 of the cells
exhibited cytopathic effect. Progeny virus
remains associated with cell debris. Cells were
scraped off the dish and the cells and media
centrifuged _at_ 700 x g for 5 min. The cell pellet
was suspended in 1/20th of its original volume
in spent media containing 10 glycerol,
suspended and subjected to 3 cycles of
freeze-thawing to release the virions from the
cell debris. Virus was dispensed in 50 µl
aliquots and frozen. Virus was assayed on 293
cells by standard plaque assay. Briefly, 35 mM
cluster dishes were seeded with 293 cells at
least 24 hrs prior to the assay. When monolayers
were 80 confluent, medium was removed and 200 µl
of serial 10-fold dilutions of the virus added.
Dishes were rocked every 15 min for 1 hr.
Inoculum was removed and agarose overlay added.
Dishes were placed at 40C for 10 min., then
incubated 6 days, 370C, 100 humidity in 5 CO2.
Overlay was prepared by making 2 x growth medium,
warming it to 370C and mixing it with 370C 2
aqueous SeaPlaque low gelling temp agarose (FMC
Corp. Rockland ME), mixing, incubating _at_370C 10
min, adding 3ml overlay/well. After incubating 6
days, 2 ml additional overlay containing 50 µg/ml
neutral red was added and dishes incubated
overnight. Plaques appeared as light areas on a
red background. Values were reported as plaque
forming units (pfu)/ml. Each value was the mean
of at least two dishes. Transduction
experiments were performed in 96-well trays.
Twenty-four hrs prior to transduction 5 x 104 MDA
cells were plated per well. Medium was removed
and replaced with 20 µl of control medium or
medium with diluted virus. Cells were transduced
with a multiplicity of infection (MOI) of virus
of 50 (i.e., 50 virions per cell). Dishes were
rocked every 15 min., for 1 hr, incubated 3
additional hrs, then 200 µl of medium or medium
and test chemicals were added to each well,
followed by 48 hr incubation. Plates were washed
2 x with PBS ph 7.4, decanted and 25 µl Cell
Culture Lysis Reagent 25 mM Tris-phosphate, ph
7.8, 2 mM DTT, 2 mM 1,2-diaminocyclohexane-N,N,N,
N tetraacetic acid, 10 glycerol, 1 Triton
X-100 (Promega Corp., Madison WI) added,
incubated 30 min., or until cells lysed. Plates
were either frozen at -80oC, or assayed
immediately for luciferase activity. Each well
received 25 µl reaction buffer ( 25 mM
glycylglycine, 15 mM MgCl2, 5 mM ATP, 0.1 mg/ml
BSA, pH 7.8), followed by 25 µl 1mM D-luciferin 5
seconds later. Luciferase activity was
quantitated in a MLX microtiter plate luminometer
(Dynex Tech, Chantilly VA) and data expressed in
relative light units (RLU). All chemicals were
purchased from Sigma (St. Louis, MO) unless
stated otherwise. Hydroxyflutamide (OHF) was
provided by R.O. Neri Schering Corp. (Bloomfield
NJ). OH-DDE was purchased from SPECS and BioSPECS
B.V. (Rijswijk, Netherlands). Vinclozolin
metabolite, M2, was obtained from BASF Ag and
metabolite, M1, was synthesized from vinclozolin
and purified as previously described (Kelce et
al., 1994 TAP 126276-285). Synthesis of the
methoxychlor metabolite, HPTE, was previously
described (Waller et al., 1996, TAP 137219-227).
1.0
14.0
M1
M1 0.1nM DHT
12.0
M2
M2 0.1nM DHT
10.0
0.1
0.1 nM
DHT
ET-OH
.05 uM
0.2 uM
0.5 uM
10 uM
DHT
1 uM
8.0
E2
1.0
3.5
6.0
8.4
39.8
26.5
6.3
2.9
3.5
4.7
25.4
E2 1uM ICI
Relative Light Units
E2 1uM OHF
1.0
1.3
2.6
37.2
6.0
4.0
Dexamethasone-induced Luciferase Expression in
Adenovirus Transduced
MDA-453 Cells
1000.0
2.0
Dex
Dex 0.1 nM DHT
0.0
0.1 nM
0.1 nM
ET-OH
.05 uM
0.2 uM
0.5 uM
1.0 uM
10 uM
DHT 1
100.0
DHT
uM OHF
0.6
0.6
0.5
0.6
0.7
0.9
10.7
1.1
M1
9.5
3.5
2.5
1.6
0.6
M1 0.1nM DHT
0.7
1.2
0.7
0.9
2.4
M2
Relative Light Units
6.3
3.0
1.0
1.0
2.4
10.0
M2 0.1nM DHT
1.0
0.1
DHT
.01 nM
0.1 nM
1 nM
10 nM
100 nM
ET-OH
DHT
1 uM
Dex
Dex
Dex
Dex
Dex
OHF
Dex
0.6
1.0
1.3
2.3
38.5
124.2
13.3
1.1
Dex 0.1 nM DHT
8.8
8.8
14.5
41.4
115.0