Taylor et al. BMC Cancer 2010, 10:541 http://www.biomedcentral.com/1471-2407/10/541 after carcinogen administration the animals received sc an estradiol pellet (0.18 mg/pellet: Cat No: SE-121, Innovative Research of America, Saratoga, FL), aiming to further stimulate PEC proliferation, as was reported previously [23,24]. However, in the second experiment, within four weeks, about 50% of p27+/- and p27-/- ani- mals developed severe prostate hypertrophy, leading to almost complete obliteration of urethra, causing urine retention in the bladder and death by kidney failure. Carcinogen N-Methyl-N-Nitrosourea (MNU), was obtained from Ash Stevens Inc. (Detroit, MI), dissolved in sterile acidified saline (pH 5.0), and injected ip at 30 mg/kg body weight six days after sc testosterone treatment or implantation of testosterone pellets. At that time, the number of prolif- erating PEC sharply increased (see Table 1), suggesting that a high number of proliferating cells would facilitate MNU-induced prostate carcinogenesis. 9-cis retinoic acid (9cRA) was obtained from the repository of the National Cancer Institute. It was sus- pended in purified sesame oil (vehicle) and given by gavage by #20 gauge needle in 0.1 ml 6 days a week at a concentration of 30 mg/kg body weight. For the long- term chemoprevention study, 9cRA was mixed in the diet at 50 mg/kg. Histomorphology Mouse prostate consists of 3 glands: dorso-lateral (DLP), anterior (AP, coagulating gland) and ventral prostate (VP). Seminal vesicles were also macroscopically exam- ined as part of the prostate tissue complex. During autopsy all the above glands were removed, separated under a dissection microscope from the urinary bladder and urethra, fixed in 10% formalin, and embedded in par- affin [29]. DLP and AP were cut longitudinally through the urethral part of the prostate and both tissue pieces were put in the cassette with the cutting area facing down. Stepwise, 4 μm thick paraffin sections at three separate tissue levels 30 μm apart were cut. On each slide 3 tissue sections were mounted. All resulting slides were labeled consecutively. This method of embedding and Page 3 of 12 sectioning has proved to be essential for detecting small lesions and micro invasive carcinomas and accurately tracing their origin and structural characteristics. RARa and RXRa expression Both receptors were determined by immunocytochemis- try employing, respectively, anti-RARa (SC-551, Santa Cruz Biotech.) and anti-RXRa (SC-553) antibodies and ABC kit. Only cells with staining localized in the nucleus were considered positively stained. Proliferating cells In the short-term study, proliferating cells were identi- fied by BrdU-labeling. Two hrs prior to sacrifice, mice were ip injected with 5-Bromodeoxyuridine (BrdU), 50 mg/kg body. BrdU-labeled cells were detected by their corresponding antibody (Santa Cruz Biotech. Inc., CA) and ABC kit, as we reported previously [28]. In the long-term cancer prevention study, proliferating cells were identified at the end of experiment by Ki-67 anti- body (Santa Cruz Biotech. Inc.) and ABC-kit. Apoptosis Apoptotic cells were identified on parallel tissue sections by TUNEL assay, as recommended by the ApopTag in situ hybridization detection kit (Oncor, Co., Gaithersburg, MD). Tissue sections were counterstained by methyl green for visualization of tumor morphology. Senescent cells An SA-b-Gal activity assay was employed to identify senescent cells in prostate tissues and tumors [30]. Fro- zen sections (5-7 μm thick) from control and cells trea- ted with 9cRA were fixed in 3.0% formaldehyde for 5 min, washed in PBS, and stained in b-Gal (5-bromo-4- chloro-3-indolyl-b-D-galactosidase) (Sigma Chem., Co. St. Louis, MO) solution at pH 6.0 for 24 hrs at 37°C. Statistical analysis Differences in the incidence of prostate lesions among various groups were evaluated by the ANOVA test fol- lowed by the LSD test. Differences in the average Table 1 Effects of testosterone and 9cRA on cell proliferation in DLP Genotype p27+/+ p27+/- p27-/- No 5 6 6 Controls BrdU (%) 0.3 ± 0.2 0.3 ± 0.2 0.4 ± 0.2 No 6 5 6 9cRA BrdU (%) 0.3 ± 0.2 0.5 ± 0.4 0.5 ± 0.3 No 5 8 9 Testosterone BrdU (%) 0.7 ± 0.3 ** 1.5 ± 0.6* ** 1.7 ± 0.3* ** No 6 6 5 Testosterone+9cRA BrdU (%) 0.7 ± 0.3 ** 1.4 ± 0.5* ** 1.5 ± 0.3* ** Two-month-old p27+/+, p27+/-, and p27-/- mice were treated for 7 days with: placebo (sesame oil-control group); 9cRA, 30 mg/kg body weight; testosterone, 1.0 mg/kg body weight; or combination of 9cRA + testosterone. BrdU, at 50 mg/kg body weight was injected 2 hrs prior to animal’s sacrifice. BrdU-labeled cells were evaluated among epithelial cells of DLP by counting at least 500 cells per animal. Two to three levels of sectioning, 30 microns apart, were employed in some animals to reach the above number of cells. The evaluation of BrdU-labeled cells was done blindly, without knowledge of the animals’ genotype and the treatment protocol. No, number of animals in each group; *, significant difference (p < 0.05) as compared to the values in p27+/+ mice; **, significant difference (p < 0.05) as compared to control values.