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Androgen Biology Group

Androgen Receptor Pharmacology

William T. Schrader, Ph.D.
William T. Schrader, Ph.D.
Deputy Scientific Director, Principal Investigator

Tel (919) 541-3433
Fax (919) 541-5466
P.O. Box 12233
Mail Drop A3-05
Research Triangle Park, North Carolina 27709
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Research Summary

The Androgen Biology Group, established in 2003, studies the pharmacology of tissue-selective androgen receptor modulators.

The group studies the pharmacology of tissue-selective androgen receptor modulators. The methods employed involve capitalizing upon unique non-steroidal androgen agonists and antagonists that are photo-activatable by visible light. Group members use both cell- and animal-based assays to investigate the pharmacodynamics of these compounds, with particular reference to their utility to induce androgen receptor (AR)-dependent cell death.

The principal compound of interest is 1,2,3,4-tetrahydro-2,2-dimethyl-6-(trifluoromethyl)-8-pyridono[5,6-g]quinoline, or TDPQ. This compound is a potent, tissue-selective modulator of the androgen receptor. TDPQ absorbs light at 400 nm, and can elicit formation of singlet oxygens [1O2]. Singlet oxygens can in turn elicit production of other reactive oxygen species [ROS] that can cause toxic effects in cells by a number of mechanisms.

Apoptosis Induced by Light-simulated Androgen Receptor Ligand
Apoptosis Induced by Light-simulated Androgen Receptor Ligand

The group tested TDPQ’s photocytotoxicity in a live-cell assay using a fluorescence microscope with a cell-growth chamber. The AR-positive human prostate carcinoma (LNCaP) cells were used first. TDPQ caused translocation of AR to nuclei as shown by immunohistochemistry. Irradiation of the live cells in culture on the microscope stage generated a cell-killing effect that was dependent on time, light and compound concentration. Cells progressed through a permeable stage visualized via propidium iodide staining, followed subsequently by a cytolytic phase resulting in cell death. Group members are investigating the mechanism of this pathway.

AR is required to elicit the photocytotoxic effect of TDPQ. Suppression of AR expression using siRNA in LNCaP cells blocks the effect, as does simultaneous incubation of the cells with a large excess of the natural physiologic AR hormone, dihydrotestosterone. PC3, another human prostate carcinoma cell line which lacks AR. These cells are highly resistant to the TDPQ photocytotoxic effect. However, when AR is introduced into the cells by stable transfection with an AR expression plasmid, the resulting AR-containing cells are ten times more sensitive to killing by TDPQ than the original PC3 cells. These findings demonstrate that TDPQ acts as a photosensitizer that acts in an AR-dependent manner to induce cell killing.

Figure 1: Chemical structure of the non-steroidal androgen receptor (AR) modulator TDPQ.  Its absorbance maximum (380 nm) and strong fluorescence emission maximum at 460 nm allowed construction of a customized filter set for the Zeiss Axiovert microscope. Upon irradiation at 405 nm the compound also exhibits rearrangement to an excited triplet state, able to react with O2 to produce singlet oxygen with modest yield (7%) as evidenced by a characteristic singlet-oxygen emission at 1270 nm.
Figure 1: Chemical structure of the non-steroidal androgen receptor (AR) modulator "TDPQ". This compound is a highly selective androgen receptor. Click for larger view

The group is also studying the cell death pathway. Death by necrosis is characterized by bloating of the cells and nuclei, followed by disintegration of the cell membrane. Programmed cell death, termed apoptosis, takes place by a series of discrete enzymatic and osmotic steps. ROS have been slow in other systems to induce cell death via this pathway.

The group is testing the mechanism of TDPQ-induced cell death using molecular probes (propidium iodide, Hoechst 33342 and annexin V/FITC conjugate) to quantify cell death parameters. Cells are treated with TDPQ in doses from 0 to 3 µM and then irradiated for 3 min at λ=405 nm using irradiation doses from 0.1 to 1 kJ/cm2. Control groups consist of cells that were not exposed to either the photosensitizer or UV irradiation, or to either agent alone. Neither the photosensitizer alone nor the maximal irradiation dose of 1kJ/cm2 alone has a cell killing effect. However, cell death is induced by light irradiation in the presence of TDPQ in a time- and concentration-dependent manner.

Figure 2: Human LNCaP prostate tumor cells were grown in 8-well microscope slides on an incubated 37C chamber atop a Zeiss Axiovert microscope. Confluent cells were treated with various concentrations of the AR modulator TDPQ and with UV light (405 nm) and then incubated for up to 24 hours. Then cells were stained with the permeable dye Hoechst 33342 to stain all nuclei, and simultaneously stained with propidium iodide (PI) to stain cells permeabilized by the rapid cell membrane changes associated with the onset of apoptosis. Top Row: Optimal irradiation time was determined by adding 30 µM TDPQ for 1 hour and then irradiating for up to 180 seconds. Cells were cultured for 4 additional hours and then stained with Hoechst and PI. Note that background apoptosis rates (about 2%) were not affected by TDPQ in the absence of irradiation. Bottom Row: Concentration curve for apoptosis induction by TDPQ. Cells were treated with the indicated concentrations of TDPQ and irradiated 1 hour later with UV, and cultured for 24 hours prior to staining. Cells progress from being propidium iodide-resistant to staining pink (apoptosis under way) to cell death and loss from the slide in the irradiated area (shown by the white circles). At the highest dose the cell death induction proceeds concentrically into the non-irradiated region of the culture.
Figure 2: Cells progress from being propidium iodide-resistant to staining pink (apoptosis under way) to cell death and loss from the slide in the irradiated area (shown by the white circles). Click for larger view

Annexin V staining, TUNEL staining and the morphologic appearance of condensed nuclear DNA proves that the majority of cell death occurs due to apoptosis. The onset of apoptosis is rapid; it is observed as early as 30 minutes after irradiation. The extent of apoptosis in the irradiated portion of the culture increases with incubation time. By about 20 hours, most of the cells within the irradiated area are dead. There is a progressive, concentric invasion of the apoptotic response into the non-irradiated portion of the well. This observation is indicative of a "bystander effect," in which cell apoptosis is spread via cell contacts. This latter feature of the mechanism is under study presently.

The mechanism by which photoactivation of TDPQ bound to AR can cause apoptosis is the focus of the future work. Reactive oxygen species can cause damage to DNA, to the AR itself, or to other cell compartments including the mitochondria. Since the TDPQ-AR are located in the nucleus at the time of irradiation, the group hypothesizes that AR-directed DNA damage occurs. Efforts are under way to map the extent and site(s) of damage.

Figure 3: Androgen receptor level in cells determines senstitivity to TDPQ-induced apoptosis. After 60 min exposure to the compound and 180 seconds’ illumination the cells were grown for 24 h and then stained with Hoechst and PI. Three human prostate tumor cell lines were compared quantitatively using a Zeiss Axiovision image analysis software program. Cell Death Index is a measure of the per cent of the initial cells in the irradiated field that have either become PI-positive (early apoptosis) or have been lost from the region (due to cell death). Left panel: Dose-response curves for TDPQ- photoinduced apoptosis. These cells, expressing high levels of AR, undergo complete apoptosis-induced cell death. Potency is about 400 nM TDPQ. Right panel: PC3 cells (AR-negative) stably transfected with an empty expression plasmid containing a neomycin selection marker. Efficacy of does not exceed 25%, even at the highest dose achievable due to limited solubility of TDPQ. Potency cannot be calculated but lies far to the right of that seen for LNCaP cells. Middle panel: PC3 cells that have been stably transfected with an AR expression plasmid. The cells express about 15% the level of AR as that seen in LNCaP cells (measured by Western immunoblotting). Efficacy is restored to 100% by the AR transfection. The cells are not as sensitive to TDPQ; the potency is shifted to the right to about 2 µM.
Figure 3: Three human prostate tumor cell lines were compared quantitatively using a Zeiss Axiovision image analysis software program.

Due to the selectivity shown to kill only AR-positive cells, this photocytotoxicity method can be thought of as a targeting method for causing death of certain unwanted AR-positive cells while sparing their AR-negative neighbors. For example the TDPQ-AR apoptotic effect has potential therapeutic implications for the treatment of androgenetic diseases of the skin (acne, male-pattern baldness and hirsutism). The group has extended its observations to include ligands that activate other members of the nuclear hormone receptor family, thereby also extending the targeting potential to other diseases as well.

Development of Cell-Based Screens for Effects of Endocrine Disruptors in Obesity

A second research activity involves studies of the role of androgen receptor modulators as controllers of stem cell differentiation in vitro. Published evidence for androgen effects to shift stem cells towards a myoid pathway as opposed to an adipocytic pathway supports a role for endocrine disruptors as environmental factors affecting obesity in humans. The mechanism and sensitivity of this process to androgen agonists and antagonists is currently the focus of this aspect of the group’s work.

The group studies human stem cell lines, including embryonic stem cells, to determine the effects of androgen receptor modulators on proliferation, differentiation and induction of multiple cell lineages. The effects of androgens (testosterone and dihydrotestosterone; DHT) are chiefly meditated via AR, which plays an essential role in a variety of biological processes, including energy expenditure, metabolism and control of body composition. In humans androgens cause two effects on body composition: (a) an increase in muscle mass and strength and (b) a decrease in fat mass. Similarly, genetically-modified AR knockout mice exhibit late onset of obesity, suggesting that AR might serve as a regulator of adipogenesis and myogenesis. These observations suggest that utilization of the AR system may provide promising approaches in the current fight against the national epidemic of obesity. Androgen receptor agonists in the environment, for example, may predispose differentiation of stem cells towards muscle cell lineage, whereas AR antagonists may direct stem cells away from muscle and towards the fat cell phenotype.

Figure 4: Development of an assay for effects of androgen receptor (AR) modulators on stem cell differentiation. Mouse C3H 10T1/2 cells were treated for 72h with 5-azacytidine (20 µM) to induce differentiation into fat or muscle cells. Cells were re-plated and treated with vehicle, AR agonist dihydrotestosterone (DHT) or AR antagonist flutamide twice weekly for a period of seven days in DMEM media containing 10% fetal bovine serum. Top Row (40x objective): Differentiation to adipocytes was observed using the lipid-specific stain Oil Red O. Lipids are stained red and nuclei are blue. Bottom row (10x objective): Differentiation to myocytes and myotubes was observed using Massons’ Trichrome stain. Muscle cells are stained red and nuclei are black. Androgen treatment favors muscle-cell lineage while suppressing fat cell lineage. Antiandrogen flutamide has the inverse effect. Complete medium contains androgens from the serum; the flutamide effect along probably represents suppression of the effects of endogenous androgens.
Figure 4: Mouse C3H 10T1/2 cells were treated for 72h with 5-azacytidine (20 µM) to induce differentiation into fat or muscle cells.

Two different cell lines, mouse C3H 10T1/2 and immature human teratoma (NCCIT; National Cancer Center Immature Teratomas) have been tested for the effects of androgens and anti-androgens on cell differentiation and induction of multiple cell lineages. Mouse C3H 10T1/2 cells were treated with an epigenetic modifier (methylation inhibitor, 5-azacytidine; 20 µM) to induce differentiation into fat or muscle cells. Three days later, these cells were also treated with (DHT; 0- 30 nM) and/or flutamide (200 nM) twice weekly for a period of seven days. AR was detected immunocytochemically at low levels in untreated and in 5-azacytidine treated 10T1/2 cells. AR expression increased in DHT treated cells in a concentration dependent manner, while no increase was observed in the flutamide treated group. Differentiation of myocytes and formation of myotubes were observed by using Massons’ Trichrome staining and with a muscle specific antibody for the protein MyoD, whereas oil red O staining and an adipocyte specific antibody for the protein PPARγ were used for monitoring differentiation and formation of fat cells. Differentiation in these cells occurred spontaneously.

Group members are currently involved in developing homogeneous assays for cell differentiation using fluorogenic dyes. This method can be adapted to 96-well formats, thereby facilitating high-throughput screens of compounds to test for their potential to alter differentiation pathways.

Major areas of research:

  • Biology of tissue-selective androgen receptor ligands
  • Molecular pathways regulated by steroid hormones

Current projects:

  • Studying the induction of apoptosis via photosensitization of cells in an androgen-receptor dependent pathway
  • Testing the effects of androgens and anti-androgens on cell differentiation and induction in stem cells

William T. Schrader, Ph.D., head of the Androgen Biology Group, is a biochemist and molecular endocrinologist. His research interests have dealt with the structure, function and regulation of the steroid receptor superfamily. A native of Long Island, New York, he received the Ph.D. degree in biology from Johns Hopkins University in 1969, and then did postdoctoral research at Vanderbilt Medical School before joining the faculty at Baylor College of Medicine in 1972.  He was appointed Professor of Cell Biology in 1985 and became Assistant Dean of the Graduate School in 1991.  He joined Ligand Pharmaceuticals in 1995 as Vice President for Endocrine Research where he directed drug discovery in the areas of female and male sex hormone receptor modulators. Several of these drugs have advanced into human clinical trials. In 2000 he co-founded XenoPharm, Inc. and served as the company’s Chief Scientific Officer and Vice President for Research. The company’s technical platform commercialized assays based upon proteins of the liver and intestine that sense the presence of foreign small molecules, including drugs and environmental substances.

Schrader joined the National Institute of Environmental Health Sciences in 2003 as Deputy Scientific Director. In that role he deals extensively with postdoctoral training and career development. His research laboratory studies the mechanism of action of tissue-selective nonsteroidal androgen receptor modulators and other substances that affect sex hormone developmental pathways. He has served on numerous editorial boards, study sections and advisory panels for educational, governmental and for-profit organizations.

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Last Reviewed: May 14, 2007