Taylor et al. BMC Cancer 2010, 10:541 http://www.biomedcentral.com/1471-2407/10/541 Table 2 Alteration in body weight and in DLP weight in the course of prostate carcinogenesis Genotype p27+/+ p27+/- p27-/- 8 10 12 Body wt: (g) Placebo 30.8 ± 4.3* 35.8 ± 5.1 41.4 ± 5.6* 10 12 10 Body wt. (g) 9cRA 27.2 ± 5.4* 31.3 ± 5.6 35.5 ± 6.4* DLP-(mg) Placebo 48.3 ± 13.1** 67.2 ± 14.5 97.6 ± 21.0** x Page 5 of 12 DLP-(mg) 9cRA 45.6 ± 7.7** 60.2 ± 10.5 80.5 ± 18.6** x These are the final measurements of body weight (g) at the end of experiment when the animals were sacrificed. DLP was isolated at autopsy, measured on an analytical balance and then frozen or embedded in 10% neutral formalin. Body and DLP weights were higher in p27-/- as compared to p27+/+ mice; *, p < 0.01; **, 0.001. 9cRA also decreased DLP weight in p27-/- mice (x) as compared to placebo-treated mice (p < 0.02). chemopreventive efficacy of 9cRA. In previous studies, others [23] and we [24] have shown that the combina- tion of carcinogen + testosterone + estradiol stimulates prostate carcinogenesis in rats. For this study, a testos- terone pellet was first sc implanted to simulate cell pro- liferation and 6 days later the animals were injected with MNU to initiate prostate carcinogenesis. Four days after carcinogen administration an estradiol pellet was sc implanted. Both testosterone and estradiol pellets were designed to release hormones within 60 days, with a second and third pellet projected to be implanted after those initial 60 days. However, testosterone + estradiol treatment induced severe prostate hypertrophy, predo- minantly in p27-/- and p27+/- mice, with complete blockage of urethra that caused urine retention in the bladder, and about 50% of animals died within 4-6 weeks due to kidney failure and intoxication. Therefore, the number of animals in individual groups at the end of the experiment (Table 4) is relatively small, as com- pared to 20 animals per group at the beginning of the experiment. The experiment continued with surviving animals, and no second or third pellets were implanted. As shown in Table 4, PIN was identified in all p27 gen- otypes, with the lowest value in p27+/+ (1 in 15 Table 3 9cRA inhibits MNU-induced prostate carcinogenesis in p27 deficient mice PIN Genotype Animals 9cRA Incidence Frequency No P Tumors P DO p27+/+ p27+/+ p27+/- p27+/- p27-/- p27-/- 18 17 16 15 15 15 0 + 0 + 0 + 11 0 60 44 70 53 0.16 ± 0.5 0 0 0 0 1.5 ± 0.5* 3 1.1 ± 0.6 0.05 2 2.0 ± 0.4* 3 1.4 ± 0.5 0.005 4 0 0 4 4 6 6 Animals were treated with MNU, 30 mg/kg body weight at the age of 2 months and followed until sacrifice between 7-12 months of age. PIN were identified in DLP by cutting longitudinally through the urethral part of the prostate. H&E staining was used for identification of PIN. Stepwise, 4 μm thick paraffin sections at three separate tissue levels 30 μm apart were cut and the number of PIN per prostate determined. Incidence, percentage of animals with PIN per group; frequency, number of PIN per animal. PIN were determined in various groups without knowledge of the treatment protocol. (P), prostate; (DO), distant organs; *, significant difference (p < 0.01) in the multiplicity of PIN between p27+/- and p27-/- mice. animals), and its incidence and frequency increased in p27+/- (40% for incidence and 0.7 ± 0.3 for frequency) and progressed further in p27-/- mice (80% for inci- dence and 2.3 ± 1.2 (p < 0.05) for frequency). Tumors localized in prostate and distant organs (DO) were also detected in p27+/- (No: 7) and in p27-/- (No: 10) mice. Treatment of animals with 9cRA was less efficacious than in animals not undergoing hormone stimulation. Thus, the incidence of PIN was similar in placebo- and 9cRA-treated p27+/+ and p27-/- mice, 13% in both pla- cebo groups and 40% and 60% in treated animals, respectively (p < 0.5); even in 9cRA-treated mice a ten- dency for increase of PIN multiplicity was observed, ris- ing from 0.7 ± 0.3 in placebo to 1.5 ± 1.1 (p < 0.1). In p27-/- mice hormone stimulation for 60 days further increased PIN multiplicity (2.3 ± 1.2 per animal) as compared to p27+/+ and p27+/- mice, but 9cRA was again not efficacious in suppressing the neoplastic pro- cess (Table 4). 9cRA inhibits prostate cell proliferation and induces cellular senescence Since 9cRA can modulate cell growth by retinoid recep- tor dependent and independent mechanisms, RARa and RXRa were also determined by ICH. These two recep- tors are chief targets of 9cRA in epithelial cells [22]. As shown in Fig 1G, RARa is expressed in prostate epithe- lial cells of non-PIN (right gland), as well as in the papillary structures of PIN (left gland, arrow); note the nuclear RARa location. RXRa was also detected in almost all PIN cells (Fig. 1H, arrow). An attempt was made to compare the percentage of RARa and RXRa in placebo- and 9cRA-treated animals, but no difference was found (data not presented), suggesting a receptor independent mechanism of cell growth inhibition. To further understand whether 9cRA suppresses prostate carcinogenesis by inhibiting cell proliferation or/and by inducing apoptosis and/or cellular senescence (CS), mice treated with MNU and MNU + 9cRA (Table 3) were sacrificed at the end of the experiment, DLP and AP were frozen on dry ice, and then frozen tissue sec- tions were prepared and treated with SA-b-Gal kit for identification of senescent cells. Cell proliferation was determined on frozen sections by Ki-67 antibody