Cell lines, antibodies, and reagents.
C42LucBAD cells were generated by transfecting C42 cells with WT BAD (HA-BAD-pTRE2hygro) and firefly luciferase (PGL4.13). C42LucPKI cells were developed by cotransfecting C42tet-on cells with PKI-GFP chimera (PKI-GFP-pTRE-tight) and firefly luciferase (PGL4.14 hygro). C42LucBAD1SA
cells were developed by cotransfecting C42tet-on cells with mutant BAD1SA
-pTRE-tight), which has a mutated phosphorylation site at S112, and with firefly luciferase (PGL4.14 hygro). Transfections were performed at 60%–70% confluence using Lipofectamine 2000 (Invitrogen) according to the manufacturer’s recommendations. All cell lines were maintained in RPMI 1640 with 10% fetal bovine serum in 5% CO2
at 37°C. Selections of stable clones were carried out in the presence of G418 (600 μg/ml) and hygromycin (150 μg/ml). After a 3-week selection period, macroscopically visible colonies were picked based on luminescence (luciferase expression). Cells were then expanded and analyzed for doxycycline-inducible expression of HA-BAD, PKI-GFP, and HA-BAD1SA
. A2870 ovarian cancer cells, which do not express ADRB2, were provided by A. Sood (University of Texas MD Anderson Cancer Center, Houston, Texas, USA).
Antibodies were obtained from the following sources: BAD, pBADS112, pAKT308, pAKTS473, pCREBS133, and cleaved caspase-3 from Cell Signaling Technology; mouse monoclonal antibody to β-actin from Sigma-Aldrich; goat polyclonal antibody to cleaved PARP from R&D Systems; anti-ADRB2 antibody from Olomone Labs; mouse monoclonal antibody to c-Myc (9E10) and goat polyclonal antibody to probasin (R-15) from Santa Cruz Biotechnology; rabbit polyclonal antibody to FVIII-RA (catalog no. 18-0018) from Zymed; and secondary horseradish peroxidase–conjugated antibodies used for Western blots from Amersham Biosciences. Protein G–agarose beads and adrenaline were obtained from Calbiochem. ZSTK474 was provided by Zenyaku-Kogyo Co. Ltd. G418 and hygromycin B were from Clontech. Bicalutamide (Casodex) was bought from Santa Cruz Biotechnology. All other chemicals and reagents, unless otherwise specified, were purchased from Sigma-Aldrich. Tissue culture reagents were purchased from Invitrogen.
Mouse xenograft model and acute stress.
6-week-old male athymic nude mice (BALB/c genetic background) were obtained from the National Cancer Institute. Mice were maintained under pathogen-free conditions and provided with sterile food and water ad libitum. Human tumor xenografts were generated by subcutaneously inoculating prostate cancer cell lines (C42LucBAD, C42LucPKI, and C42LucBAD1SA) into nude mice. Each mouse received 4 subcutaneous injections of 2 × 106 cells with BD Matrigel matrix High Concentration (BD Biosciences). Injections were made using an insulin syringe and a 27-gauge needle at 4 locations: left and right shoulder and left and right flank. Injections at 4 locations ensured that each mouse would develop xenograft tumors, thus reducing the number of mice required for the experiments. When the largest tumor was approximately 100 mm3 in size, HA-BAD, PKI-GFP, or BAD1SA expression was induced by addition of doxycycline (1 mg/ml) to the drinking water 24 hours before the experiments. Mice were randomly assigned to experimental groups (DSMO; ZSTK474; ZSTK474 followed by immobilization stress; ZSTK474 and epinephrine; ZSTK474 and ICI118,551, followed by immobilization stress; and ZSTK474, ICI118,551, and epinephrine). The next day, xenograft tumors (1 tumor per mouse) were injected once with the PI3K inhibitor ZSTK474 (5 mM, 40 μl; 0.8 mg/kg) or vehicle (DMSO); 3 hours later, mice were subjected to immobilization stress for 1 hour (acute stress) or injected with adrenaline (100 μM, 30 μl). Immobilization stress, which mimicked the presence of a natural predator without possibility of escape, was created by placing mice into a 50-ml conical vial with openings for breathing. Vials with mice were placed for 1 hour in a plastic box that contained tissue impregnated with fox urine (Chagnon’s Trapping Supply). The ADRB2 antagonist ICI118,551 (25 μM, 30 μl) was given intraperitoneally 30 minutes before immobilization stress or adrenaline injection. To avoid unintended stress, mice were handled with extra care. Manipulations that could cause distress (e.g., injections and blood sampling) were conducted under light isoflurane anesthesia.
Xenograft tumors were monitored by noninvasive optical imaging on an IVIS100 imaging station (Caliper Life Sciences). Animals were immobilized by gas anesthesia with 2% isoflurane/O2. To account for background and nonspecific luminescence, mice were imaged before injection of luciferin. Animals were injected with 100 μl of the firefly luciferase substrate luciferin (3.5 mg/ml in PBS) and imaged 15 minutes later. Whole-body images were obtained and analyzed using the Living ImageH software provided with the imaging system. A grayscale photographic image and the bioluminescent color image were superimposed to provide anatomic registration of the light signal. A region of interest (ROI) was manually selected over the luminescent signal, and the intensity was recorded as photons/second within an ROI. Luminescence was measured before injection of PI3K inhibitors (0 hours) and 24, 48, and 72 hours after injection. Xenograft tumors were excised 6 hours after injection of ZSTK474 and analyzed by immunohistochemistry for expression of cleaved caspase-3 and by Western blotting for expression of BAD, PKI-GFP, and BAD1SA; inhibition of pAKT; cleavage of PARP and caspase-3; and phosphorylation of CREB and BAD. At the time of tumor excision, blood was also collected, and adrenaline levels were measured by ELISA (see below).
Mouse prostate model and chronic stress.
The transgenic Hi-Myc mice used herein, in which the prostate-specific expression of human c-myc is driven by the rat probasin promoter with androgen-responsive elements (ARR2/probasin promoter; ref. 29
), were obtained from the Mouse Repository of the National Cancer Institute Mouse Models of Human Cancer Consortium. Hemizygous Hi-Myc mice on FVB background were cross-bred with nontransgenic FVB breeders. All control mice used at each time point were from FVB littermates. To test the role of BAD phosphorylation, Hi-Myc mice were bred with BAD3SA/3SA
knockin mice (provided by M. Greenberg, Harvard Medical School, Boston, Massachusetts, USA), in which endogenous BAD is replaced by mutant BAD with S112, S136, and S155 substituted for alanines (14
). In these experiments, the second generation of Hi-MycBAD3SA/WT
mice was compared with WTBAD3SA/WT
littermates as controls for baseline measures of prostate weight and with Hi-MycBADWT/WT
littermates as controls for stress-induced reduction of apoptosis and increase in prostate weight.
12-week-old mice were subjected to immobilization stress for 1 hour for 7 consecutive days (2 times per day with 12-hour interval). To investigate the role of ADRB2 in stress effects, ICI118,551 (25 μM, 30 μl) was given intraperitoneally 30 minutes before immobilization stress. At the end of day 7, before sacrifice of animals, approximately 500 μl of blood per mouse was collected through heart puncture under deep isoflurane anesthesia; blood was stored at –80°C until analysis. After sacrifice, each mouse was weighed. Anterior prostate (AP), DLP, and ventral prostate (VP) lobes were dissected and weighed; prostate tissues were then paraffin embedded and snap frozen in liquid nitrogen.
In vivo bicalutamide injection in mouse stress model.
12-week-old mice were subjected to subcutaneous injections with either vehicle (DMSO) or bicalutamide (50 mg/kg) once a day and to immobilization stress for 1 hour (2 times per day with 12-hour interval) for 2 or 3 consecutive days. To investigate the role of ADRB2 in stress effects, ICI118,551 was given as described above. At 24 hours after the last injection, blood and prostate tissues (AP, DLP, and VP) were collected and processed as described earlier.
Blood collection from prostate cancer patients.
Blood was collected by phlebotomy twice with a 4-week interval.
Adrenaline and noradrenaline measurements.
Plasma adrenaline concentrations were measured by ELISA (mouse) or radioimmunoassay (mouse, human) using commercially available assays (BA-0100 and BA-5100; Labor Diagnostika Nord, purchased through Rocky Mountain Diagnostics). Adrenaline was first extracted using a cis-diol–specific affinity gel, acetylated to N-acyladrenaline, and then derivatized enzymatically to N-acylmetanephrine. Acylated adrenaline from the standards, controls, and samples then competed for a fixed number of antiserum binding sites, later detected by ELISA or radioimmunoassay, as described by the manufacturer.
For tissue adrenaline measurements, samples were homogenized in 0.01 N HCl in the presence of EDTA (final concentration, 1 mM) and sodium metabisulfite (final concentration, 4 mM), and adrenaline concentrations were measured by ELISA (mouse).
Tissue noradrenaline levels were quantified using HPLC (Agilent 1100 binary HPLC) tandem mass spectrometry (Waters Quattro Ultima). Frozen pulverized tissues were weighed, suspended in HPLC-grade methanol (Fisher Scientific), and homogenized by ultrasonic disruption using a Misonix 3000 tissue homogenizer. Samples were centrifuged at 15,000 g for 5 minutes at 5°C to pellet cellular debris, and 0.1 ml supernatant was then mixed 1:1 with 20 mM ammonium acetate (pH 3.5) for analysis. Chromatographic resolution of noradrenaline was achieved using a Phenomenex Synergi4 Hydro-RP (150 × 2 mm) analytical column with a linear mobile phase gradient separation method. Mass spectrometry analysis was performed using electrospray positive ionization in multiple reaction-monitoring modes.
VEGF concentrations in the mouse plasma were measured using mouse VEGF Quantikine immunoassays (R&D Systems) following the manufacturer’s instructions.
Prostate tissues were homogenized in lysis buffer containing 20 mM Tris (pH 7.4); 40 mM NaF; 2 mM EDTA; 1 mM EGTA; 1% Triton X-100; 1 mg/ml each of leupeptin, pepstatin, and aprotinin; 1 mM phenylmethylsulfonyl fluoride; 1 mM NaVO4; 50 mM β-glycerophosphate; 40 mM p-nitrophenyl phosphate; and 1 mM dithiothreitol. The lysates were cleared of insoluble material by centrifugation at 18,400 g for 10 minutes at 4°C. Tissue extracts were incubated with 6–8 μg anti-HA antibodies (12CA5) overnight at 4°C and with protein G–agarose beads (Calbiochem) for another 3 hours. Beads were washed 3 times with cell lysis buffer, and proteins were eluted with SDS sample buffer for Western blot analysis.
Western blot analysis.
Tissue or cell lysates with equal amounts of total protein were separated by PAGE and transferred to nitrocellulose membranes for analysis with appropriate antibodies. The membranes were incubated overnight at 4°C with primary antibodies against pCREB, pAKT, AKT, pBAD, BAD, cleaved caspase-3, and GFP (Cell Signaling Technology); β-actin; and c-Myc and cleaved PARP (Santa Cruz), followed by 1 hour of incubation at room temperature with secondary horseradish peroxidase–conjugated polyclonal anti-rabbit antibody from donkeys (1:5,000 dilution; Amersham), anti-mouse IgG, HRP-Linked Whole Ab from sheep (1:5,000 dilution; GE Healthcare NA931), or anti-goat IgG, HRP-Linked Whole Ab from cows. Proteins were visualized using an ECL chemiluminescence detection system, following the manufacturer’s protocol and Hyperfilm ECL (Amersham). After staining, nitrocellulose blots were stripped and reprobed with loading control antibodies (β-actin, α-tubulin, AKT, and BAD) for comparison and normalization. For quantification of Western blots, fluorescent secondary antibodies were used, and signal was quantified on an Odyssey imaging system.
Antibody staining was performed on histological sections of formalin-fixed prostate tumor xenografts and mouse prostate lobes. Cleaved caspase-3 staining was performed with an anti–cleaved caspase-3 primary antibody (1:1,000 dilution; catalog 9661; Cell Signaling Technology) that specifically recognizes the large fragment (17 kDa) of the active protein but not full-length caspase-3, followed by a biotinylated anti-rabbit secondary antibody and streptavidin alkaline phosphatase (Super Sensitive Link-Label IHC Detection Systems; Bio-Genex); sections were then visualized with Vector Red Substrate (SK-5100; Vector Laboratories) and counterstained with hematoxylin. Similarly, Ki-67 immunostaining was performed with polyclonal anti–Ki-67 (1:200 dilution; ab15580; Abcam) antibody.
Double immunofluorescence for cleaved caspase-3 and TUNEL.
To visualize the colocalization of activated caspase-3 and DNA fragmentation in C42LucBAD prostate cancer cells, C42LucBAD prostate cancer xenografts, and mouse prostates, sequential immunofluorescence for cleaved caspase-3 and TUNEL was done as described previously (60
). Briefly, cells and fresh-frozen tissue sections were fixed in 10% buffered formalin for 20 minutes and then permeabilized in 0.1% sodium citrate with 0.1% NP-40 at 4°C for 2 minutes, followed by blocking for 30 minutes with 2.5% goat serum in PBST (PBS plus 0.1% Tween 20) at 37°C. Primary antibody to cleaved caspase-3 (1:300 dilution in PBST with 2.5% goat serum; Cell Signaling Technology) was added for 3 hours at room temperature. After washing 3 times in PBST, secondary antibody conjugated with Texas Red (1:300 dilution in PBST plus 2.5% goat serum) was added for 90 minutes at room temperature. After washing 3 times in PBST, TUNEL reaction was done as described in the manual for In Situ Cell Death Detection Kit, POD (Hoffmann-La Roche Ltd.).
Determination of apoptotic and proliferative indices.
For this study, activated cleaved caspase-3 labeling was used to identify apoptotic cells. The number of cleaved caspase-3–labeled cells in immunostained sections was counted relative to the total number of glandular epithelial cells present in whole DLP sections. Digital copies of the entire prostate were created automatically from the cleaved caspase-3–immunostained glass slides by the Aperio ScanScope CS with objective ×20. Individual images of DLP were then exported in Adobe Photoshop CS, and nonglandular portions of the DLP were cropped. The total number of DLP epithelial cells was enumerated in all glands using Image-Pro Plus 4.5 software (Image Processing Solutions). Numbers of apoptotic cells were then counted in every gland from the imaged DLP. The apoptotic index for each DLP lobe was expressed as the number of apoptotic cells per 100 cells in all glands (glands with normal epithelium and PIN). The Ki-67 proliferative index was determined similarly.
PIN measurements in Hi-Myc mice.
Hi-Myc mice develop mouse PIN followed by prostatic adenocarcinoma as a result of MYC overexpression in the mouse prostate. Histologically, mouse PIN is characterized by enlargement of nuclei, prominence of nucleoli, and epithelial cell proliferation; crowding results in a pseudo-multilayer appearance (overlapping of cells), with normal architecture of the glands (Supplemental Figure 2E). PIN area was measured in DLP of intact and chronically stressed mice. H&E-stained slides were digitally scanned, exported, and cropped as described above. PIN and total glandular areas were measured in each scanned DLP, and PIN results were expressed as a percentage of total DLP glandular area.
In vivo MRI for tumor volume measurements.
Each animal was anesthetized in an induction chamber filled with a mixture of isoflurane (2%) and oxygen (2 l/min) and continued to receive isoflurane and oxygen during the procedure; typical levels during scanning were 1.5% and 1 l/min. A respiration pillow was placed over the animal’s abdomen to monitor respiration rate and to facilitate triggered acquisition. Thermostatically controlled warm air was blown into the bore of the magnet to keep the animal’s skin temperature above 35°C. The animal was placed in the center of the 7T MRI magnet in a 50-mm-inside-diameter quadrature RF coil (Doty Scientific). Whole-body T2-weighted images were acquired using low 300.2-Mhz radio waves. The entire imaging data acquisition procedure took about 15 minutes per mouse. A 3-plane localizer Rapid Acquisition with Relaxation Enhancement (RARE) pulse sequence was used with the following parameters: TR, 2100 ms; TE, 36 ms; matrix, 256 × 256; FOV, 4 cm; slice thickness, 2.0 mm; NEX, 1; giving an acquisition time of 50 seconds. The 3-plane localizer was used to plan the geometry for the high-resolution tumor volume scan. The high-resolution scan was performed using a RARE pulse sequence as well as axial slices centered on the tumor, with the following parameters: TR, 3,300 ms; TE, 45 ms; FOV, 3.0 cm; slice thickness, 1.0 mm; matrix, 256 × 256; NEX, 2; giving an acquisition time of 10 minutes. At the end of the procedure, each mouse was placed on a warming pad until it regained consciousness. Images were analyzed using ImageJ software to measure the total tumor volume in each mouse.
Total PSA electrochemiluminescence immunoassay.
Total PSA was assayed according to the manufacturer’s protocol, using a Roche Elecsys 2010 Chemistry Analyzer (Hoffmann-La Roche Ltd.) (61
). Each sample was assayed in triplicate.
Analysis of microvessel density (MVD) in mouse prostate tissue sections.
FVIII-RA expression was detected immunohistochemically using rabbit polyclonal antibody (Zymed FVIII-RA antibody) provided by L. Metheny-Barlow (Wake Forest University School of Medicine) on histological sections of formalin-fixed mouse prostate lobes. To quantify MVD, we counted total number of microvessels (clusters of endothelial cells positive for FVIII staining with central lumen were considered to be individual vessels) from digital copies of the entire prostate. MVD was then expressed as number of microvessels per square millimeter of prostate tissue section.
To compare luminescence between mouse groups in Figures and , 2-way repeated-measures ANOVA models were fit with the individual animal treated as a random effect in the model. In these models, time and group were considered as fixed effects. For each model, the group by time interaction was first examined to determine whether differences between groups were consistent over the time periods. If this interaction was nonsignificant, then the model was refit without the interaction term, and the group effect was examined, adjusting for the time of the measurement. Groups were compared using this approach. PROC MIXE in SAS version 9.2 was used to fit these models.
To determine whether differences between data sets in Figure – and Supplemental Figures 6 and 8–10 were statistically significant, Student’s t test analysis (2-tailed distribution; 2-sample unequal variance) was performed using Microsoft Excel software. A P value less than 0.03 was considered significant. Error bars show SD from the average of at least 3 samples.
All animal studies were conducted according to a protocol approved by the Institutional Animal Care and Use Committee of Wake Forest School of Medicine and conformed to the NIH Guide for the Care and Use of Laboratory Animals. For studies in prostate cancer patients, all participants provided informed consent, and the protocol was approved by the Institutional Review Board of Wake Forest School of Medicine.