Tuesday, January 23, 2007

Screening for Androgens- part 3

Screening for AR activity using Gene expression assays

*Assay involves transient 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.
· KB2 assay (Vickie S. Wilson; 2002: Toxicological Sciences 66, 69-81)
· Describe the effects of androgens in a stably transformed cell line that they developed (MDA-KB2), which expresses the human AR (hAR) and an AR-responsive promoter linked to a luciferase reporter gene (MMTV-luc).
· Main advantage of KB2 assay -- it employs a genetically modified cell line, which eliminates the effort and inherent variability associated with repeated transient transfections.
· The breast cancer cell line, MDA-MB-453, was stably transformed with the MMTV.luciferase.neo reporter gene construct.
· Since both GR and AR are present in the MDA-MB-453 cells, and both receptors can act through the MMTV promoter, compounds that act through either AR or GR activate the MMTV luciferase reporter.
· AR agonists such as dihydrotestosterone (DHT), and GR agonists such as dexamethasone (DEX), corticosterone, and aldosterone induce luciferase expression at appropriate concentrations.

· DHT consistently produced 3–9-fold induction at concentrations from 0.1 to 10 nM.
· To distinguish AR- from GR-mediated ligands, chemicals were assayed concurrently with the antiandrogen, hydroxyflutamide (OHF), which blocks AR- but not GR-mediated responses.
· These cells are relatively easy to culture and maintain.
· Responsiveness was monitored over time and was stable for more than 80 passages.
· Some advantages -- relatively rapid (2 days), eliminates the need for transfection, can be conducted in a 96-well plate format, produces consistent reproducible results

* Construction and verification of reporter plasmid containing MMTV. neo.luc-- The neomycin gene was removed from pcDneo and inserted into pMMTV.luc

* Transformation, isolation, and screening of stable clones.
MDA-MB-453 cells (ATCC No. HTB 131) used for transformation.
Cells were seeded at 2 x 105 cells per 60-mm culture dish in maintenance medium, then transformed using Fugene 6 (Roche) per manufacturer's protocol, with 5 µg pMMTV.luc.neo per dish.
To screen clones for responsiveness to an androgen agonist, cells grown from individual colonies were plated at 1 x 104 cells per well in 96-well luminometer plates, allowed to attach. When cells were attached, the medium was replaced with freshly prepared dosing medium containing ethanol only (negative control), an agonist (dihydrotestosterone, DHT; 1 nM), or the agonist plus a known competitor (hydroxyflutamide, OHF; 1 M).
Cells were dosed with 100 µl of medium/well and incubated for 20–24 h, --harvested with
25 µl/well of lysis buffer and assayed for luciferase reporter activity.
Relative light units per well determined using a 96-well MLX Luminometer.
Clones with a minimum 2-fold response to 1 nM DHT were retained in culture.
The final clone (MDA-kb2) was chosen based on appropriate ligand responsiveness and genetic stability over time.

*Maintenance of cultures and transcriptional activation assays.
-MDA-kb2 cells stably transformed with the pMMTV.neo.luc reporter gene construct were maintained in L-15 media supplemented with, 100 U/ml penicillin, 100 µg/ml streptomycin, and 0.25 µg/ml amphotericin B at 37°C, without CO2.
-For experiments, cells were plated at 1 x 104 cells per well in 100 µl of medium in 96-well luminometer plates.
-When cells were attached (4–6 h), medium was removed and replaced with dosing medium. Stock solutions for each chemical were prepared at 1000x in ethanol.
- Dosing medium was prepared at the time of treatment by aliquoting 1 µl of stock solution into 1 ml of maintenance medium
-Vehicle control wells contained 100 µl medium/ well with1 µl of ethanol/ml of medium. Each plate also contained either 0.1 or 1.0 nM DHT, or DHT/plus 1 µM OHF as agonist control.
-Cells were incubated overnight at 37°C without CO2.
Luciferase activity was assayed --using an MLX microtiter plate luminometer and quantified as relative light units (RLU).
-Relative light units were converted to fold induction above the vehicle control value for each replicate for statistical analysis.
-Stability of Androgenic Response and Baseline Activity Androgen-induced luciferase activity measured over approximately 9 months of continuous culture to assess the stability and responsiveness of the MDA-kb2 clone.

-cells were assayed over more than 80 passages for their response to 1 nM DHT.

-At early passages, luciferase induction relative to solvent controls was about 10 fold.

-In continuous culture, luciferase induction dropped over time, but responsiveness stabilized at 30 to 40 passages.

-In later passages, a minimum of 5–6-fold induction in response to 1 nM DHT was consistently maintained over the remaining test period for more than 80 passages, demonstrating stable expression of the luciferase gene.
P. C. Hartig,1, Toxicological Sciences 66, 82-90 (2002)
*Attempt to deliver the genes via replication-defective adenovirus transduction
*describe the responses of several androgens in two AR-responsive in vitro assays. These assays used transduced MDA-453 and CV-1 cells, cell lines.
* Ad/mLuc7 virus used, which contains the luciferase gene regulated by the glucocorticoid-inducible hormone response element found in the mouse mammary tumor virus (MTV) LTR
*Transduction assays were performed in 96-well plates.
· Twenty-four h prior to transduction, 5 x 104 cells were plated per well.
· Medium was removed and replaced with 20 µl of control medium or medium with diluted virus.
· MDA cells (which contain endogenous AR) were transduced with Ad/mLuc7 reporter virus at a multiplicity of infection (MOI) of 50 (i.e., 50 virions per cell).
· CV-1 cells (which lack native AR) were transduced with Ad/mLuc7 at a MOI of 50 and Ad5hAR at a MOI of one.
· Dishes were rocked every 15 min for 1 h, incubated 3 additional h,
· 200 µl medium/medium + test chemicals added to each well
· followed by a 48-h incubation.
· Plates were either frozen at –80°C, or assayed immediately for luciferase activity.
· Luciferase activity was quantitated in an MLX microtiter plate luminometer, data expressed in relative light units (RLU).
· For androgen agonists, which stimulate luciferase expression, treatments compared to the media/ethanol control group
· Relative light units were converted to fold induction above the media value for each replicate

(L. G. Parks; Toxicological Sciences 62, 257-267 (2001)
COS whole cell human androgen receptor (hAR) binding assay

* In 3 blocks, COS cells (SV-40 transformed monkey kidney line ATCC # CRL-1650) were transiently transfected with hAR expression vector pCMVhAR
* COS cells were plated at 200,000 cells/well in 12 plates and transfected with 1 µg of pCMVhAR
* After a 3-h transfection period, cells were washed and incubated overnight
*Twenty-four hours later, medium replaced with 200 µl of serum-free/phenol red-free DMEM with R1881 plus 200 microliters of medium containing tast substance (400-µl incubation volume, with a concentration factor of 40x).
* Cells were incubated for 2 h with 5 nM [3H] R1881 at 37°C under an atmosphere of 5% CO2.
* Cells were washed in phosphate-buffered saline and lysed in 200 µl ZAP
(0.13 M ethylhexadecyldimethylammonium bromide with 3% glacial acetic acid).
* The lysate was added to 5 ml OPTI-fluor scintillation cocktail and radioactivity was counted using a Beckman LS 5000 TD counter
· 1 µM hydroxyflutamide was added to half of the samples to see if this potent antiandrogen would block the test substance-induced luciferase activity.
· Cells also were exposed to 1 nM DHT as a positive control.

CV-1 AR and GR 40-dependent transcriptional activation assays
*Experiments, each with several replicates, were conducted to determine induced AR- or GR-dependent gene expression in CV-1 cells (monkey kidney line, ATCC # CCL-70).
* 200,000 CV-1 cells were plated in a 60-mm dish and then transiently cotransfected with 1 µg pCMVhAR and 5 µg MMTV-luciferase reporter using 5 µl Fugene reagent in 95 µl serum-free medium as per the manufacturer's protocol.
· Twenty-four h after transfection, medium was aspirated and replaced with 2 ml of medium containing test substance.
· Cells then incubated at 37°C under 5% carbon dioxide.
· After 5 h of exposure, medium was removed, and cells were washed once with phosphate-buffered saline and harvested with 500 µl lysis buffer.
· Relative light units of 0.05-ml aliquots of lysate determined using a Monolight 2010 luminometer.

COS cell immunocytochemistry
Experiment conducted to visualize, by immunofluorescence, the ligand-induced nuclear translocation of hAR in COS cells.

· Two chamber slides were seeded with 100,000 cells/chamber in 2 ml DMEM supplemented with 10% FBS
· Cells transfected with 0.5 µg pCMVhAR
· Following transfection, 2 ml medium containing test substance, added to slides,
· incubated for 24 h at 37°C under 5% CO2.
· The next day, medium removed, cells washed once with DPBS (Dulbecco's phosphate buffered saline), allowed to dry for 45 min at room temperature, fixed for 10 min with 95% ethanol (-20°C), blocked with 5% BSA, and incubated overnight with primary AR antibody (1:1000) at 4°C.
· The following day, cells were washed once with DPBS and incubated with fluorescently labeled secondary antibody for 30 min at room temperature.
· To visualize the nuclei, cells were counter-stained with DAPI, a DNA stain, mounted with fluoromount
· slides examined using Microscope at 200 x magnification.
· The localization of the AR was classified as either perinuclear or nuclear in a blinded fashion from 10 randomly selected fields from a slide for each site.
· In another experiment, cells were exposed to 1 nM DHT, as a positive control.

Novel yeast bioassay system for detection of androgenic compounds.
Lee HJ, Lee YS, Kwon HB, Lee K.;Toxicol In Vitro. :2003 Apr;17(2):237-44.

Report the development of a rapid, simple, effective yeast detection system for androgenic compounds, based on the yeast two-hybrid protein interaction.
* A yeast strain, ARhLBD-ASC1, was established by co-transformation of yeast cells harboring a lacZ reporter plasmid with two vectors expressing each of LexA fused hinge-ligand binding domain (hLBD) of androgen receptor (AR) and B42 fused ASC-1 that interacts with AR-hLBD in an androgen-dependent manner.
* In this strain, androgens, but not other hormones, strongly stimulated the beta-galactosidase activity in a dose-dependent manner.

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