p ns ns ns ns Sen-% 0.8 ± 0.4 1.1 ± 0.5 0.9 0.8 ± 0.4 2.7 ± 1.3 1.0 ± 0.5 4.8 ± 1.2 1.2 ± 0.4 2.5 ± 1.3 3.2 ± 0.7 6.8 ± 2.2 Page 9 of 12 p 0.001 0.001 0.001 0.001 p27+/+ p27+/+ p27+/+ p27+/+ p27+/- p27+/- p27+/- p27+/- p27-/- p27-/- p27-/- p27-/- 5 6 5 5 6 6 5 6 6 5 6 5 PEC PEC PIN PIN PEC PEC PIN PIN PEC PEC PIN PIN 0 + 0 + 0 + 0 + 0 + 0 + Ki-67% 0.8 ± 0.3* 0.6 ± 0.4 1.2 1.4 ± 0.4* ** 1.2 ± 0.5 2.6 ± 0.4 1.5 ± 0.5 1.7 ± 0.5* ** 1.6+0.7 4.6 ± 0.4 3.5 ± 0.5 p ns 0.01 ns 0.001 ns 0.001 Apo-% 0.4 ± 0.2 0.6 ± 0.2 0.3 0.3 ± 0.2 0.4 ± 0.2 0.6 ± 0.3 0.5 ± 0.4 0.3 ± 0.2 0.4 ± 0.2 0.6 ± 0.3 0.5 ± 0.4 Taylor et al. BMC Cancer 2010, 10:541 http://www.biomedcentral.com/1471-2407/10/541 Table 5 9cRA inhibits cell proliferation (Ki-67) and induces senescent phenotype in PIN Genotype Animals Cell Type 9cRA The above data were obtained from placebo- and 9cRA-treated animals at the time of their sacrifice at 9-12 months of age. PEC, prostate epithelial cells from DLP not associated with PIN; 0, Placebo-treated animals; +, animals treated with 9cRA; PIN (low and high grade) was identified in H&E stained slides. To determine the incidence (%) and frequency of PIN in DLP and AP, 4 μm thick paraffin sections were cut longitudinally through the urethral part of the prostate stepwise at three separate levels 30 μm apart. All resulting slides were labeled consecutively. Proliferating cells were detected by Ki-67 antibody (Ki-67); Cells in apoptosis (Apo-%) were detected by ApopTag kit based on the TUNEL assay; Cells in senescence (Sen-%) were identified by SA-b-Gal staining employing a kit from Cell Signaling. At least 500 PEC or PIN cells were evaluated to determine the percentage of proliferating (Ki-67-%), apoptotic (Apo-%) or cells in senescence (Sen-%); * or ** indicates significant difference (p < 0.05) in the values between corresponding groups. associated with increased risk in developing prostate cancer [16]. Therefore, temporary or continuous down- regulation of p27Kip1 expression in PEC may promote the effect of testosterone or other growth factors in sti- mulating prostate carcinogenesis. In a previous study Fero et al. [19] reported that p27Kip1 protein expression was lacking in p27-/- mice, decreased by 50% in p27+/- mice, and promoted radiation and che- mical-induced (N-Ethyl-N-Nitrosourea-ENU) carcinogen- esis. In p27-/- mice, tumors developed in the lung, intestine, pituitary, ovary, and endometrium earlier and at a higher frequency as compared to both p27+/- and p27 +/+ mice. However, in Fero’s study prostate and male reproductive organs were not examined at the tissue level (personal communication) and neither PEC proliferation was stimulated by testosterone. In another study, Di Cris- tofano et al. [15] reported on cooperation between p27Kip1 and PTEN in developing PIN and prostate cancer. PTEN is a tumor suppressor and its activity leads to the induc- tion of p27Kip1; therefore, the lack or down-regulation of PTEN may suppress p27Kip1 expression and thus promote cell proliferation and carcinogenesis. Concomitant inacti- vation of one PTEN allele and one or both p27 alleles in transgenic mice accelerates the development and pro- gression of prostate and other malignancies [16,17]. We did not use PTEN+/-, p27-/- mice in this study because tumors in the prostate and other organs develop very early, within 3 months after birth, and most animals die at the age of 16-20 weeks. This very aggressive neoplas- tic process is difficult to modulate with retinoids and other cancer prevention agents. We found that the pro- gressive decrease of p27Kip1 expression in p27+/- and further in p27-/- mice promotes MNU-induced prostate carcinogenesis (Table 3). It appears that there is a negative correlation between the level of p27Kip1 expression and the incidence and multiplicity of PIN and prostate tumors. Since most animals were sacrificed between 9 and 12 months of age, this time period is probably not sufficient for PEC in p27+/+ mice to transform in PIN and progress in invasive carcinomas. For instance, studies on rats trea- ted with MNU have shown that prostate pre-malignant lesions and carcinomas occur 12 to 18 months after carci- nogen administration [23,24]. To accelerate prostate carci- nogenesis, we treated p27+/+, p27+/- and p27-/- mice with MNU and stimulated PEC proliferation by testoster- one + estradiol releasing pellets (Table 4). Previous studies, including ours, have shown that testosterone + estradiol stimulate PEC proliferation [35] and carcinogenesis in rats [23,24]. However, because of the severe prostate hypertro- phy with complete urethra blockage in some animals and high death rate, the study continued with surviving ani- mals only. Hormone stimulation was more efficacious in inducing PIN and tumors in p27+/- and p27-/- mice than in p27+/+ mice, suggesting that deregulation of p27Kip1 expression in PIN may promote hormone mediated pros- tate carcinogenesis in men. The data for multiplicity of PIN in Table 3, where MNU was only used to induce prostate carcinogenesis, are close to those in Table 4, where MNU + testosterone + estradiol were employed. This can be explained by termination of hormone stimula- tion which occurs 60 days after pellet implantation and which may cause cell death and thereby eliminate trans- formed PEC and their potential progression towards PIN and tumors. What is most interesting from this study was that 9cRA suppressed MNU-induced prostate carcinogenesis