Taylor et al. BMC Cancer 2010, 10:541 http://www.biomedcentral.com/1471-2407/10/541 Table 4 Incidence and frequency of PIN and tumors in mice treated with MNU + testosterone + estradiol Genotype 9cRA Animals-No PIN P Tumors* No at at Incidence Frequency start end p27+/+ p27+/+ p27+/- p27+/- p27-/- p27-/- 0 + 0 + 0 + 20 20 20 20 20 20 15 15 10 10 10 8 13 13 40 60 80 87 0.13 ± 0.3 0.13 ± 0.3 ns 0.7 ± 0.3 1.3 ± 1.1 ns 2.3 ± 1.2 1.7 ± 1.0 ns 2 1 4 3 5 5 Animals were implanted with a testosterone pellet at the age of 2 months and 6 days later MNU was injected ip to initiate the neoplastic process. Four days after carcinogen administration an estradiol pellet was sc implanted. %, incidence of tumors among control and treated animals; No: multiplicity of PIN per treated animal; ns, not significant (p > 0.05). To determine the incidence (%) and frequency of PIN in DLP, 4 μm thick longitudinally paraffin sections through the urethral part of the prostate were cut stepwise at three separate levels 30 μm apart. (Fig. 2A and 2B). The values of Ki-67 positive cells not associated with PIN in p27+/- and p27-/- control (pla- cebo-treated) mice are close, with a lack of statistical difference between them: 1.4 ± 0.3% vs. 1.7 ± 0.8% (p > 0.5), respectively (Table 4). In PIN of p27-/- mice, Ki-67 values are much higher as compared to those of p27+/- mice: 4.6 ± 1.4% vs. 2.6 ± 0.4% (p < 0.005), respectively. 9cRA was not efficacious and did not affect proliferating cells in glandular structures not associated with PIN (Fig. 2B). However, 9cRA decreased Ki-67 positive cells in PIN of both p27+/- and p27-/- mice from 2.6 ± 0.4% (placebo) to 1.5 ± 0.5% (p < 0.01) and from 4.6 ± 0.4% (placebo) to 3.5 ± 0.5% (p < 0.001), respectively. Apop- tosis was rare in both PIN and non-PIN epithelial cells of placebo-treated animals and 9cRA did not affect their number (Table 5). Surprisingly, a high percentage of senescent cells was found in PIN and non-PIN prostate epithelial cells of both placebo- and 9cRA-treated mice (Fig. 2E-H). 9cRA induced CS in non-PIN epithelial cells of both p27+/- and p27-/- mice: from 0.8 ± 0.4% in placebo to 2.7 ± 1.3% in 9cRA treated for p27+/- mice and from 1.2 ± 0.4% in placebo to 2.4 ± 0.5% in 9cRA- treated p27-/- mice. 9cRA also induced CS in PIN of both p27+/- and p27-/- mice: from 1.0 ± 0.5% in pla- cebo to 4.8 ± 1.2% in 9cRA-treated p27+/- mice (p < 0.001) and from 3.2 ± 0.7% in placebo to 6.8 ± 2.2% (p < 0.001) in 9cRA-treated p27-/- mice (Fig. 2G-H). Discussion The main goal of this study was to determine whether p27 deficiency in p27+/- and p27-/- mice may affect PEC proliferation and carcinogenesis, as well as the effi- cacy of 9cRA in suppressing the neoplastic process. p27 heterozygous (+/-) and p27 (-/-) mice were selected for the following reasons: a) the difference in p27Kip1 Page 7 of 12 expression between p27+/+, p27+/- and p27-/- mice simulates the gene’s alterations in the course of prostate cancer development in humans, as p27Kip1 is expressed in normal PEC, lacking in BPH, and differentially expressed in malignant tumors [4,8]; b) p27-/- mice develop predominantly hyperplastic and premalignant (PIN) lesions, which are much easier to suppress by can- cer preventive agents, like retinoids, than aggressively growing carcinomas such as those in PTEN mice [15-17]; c) previous studies have shown that p27 haplo- insufficiency promotes carcinogen- and radiation- induced tumors in various organs [19,20]; d) retinoids are efficacious inhibitors of prostate carcinogenesis in animal models [23,24]; and e) p27Kip1 is involved in mediating the effect of retinoids on PEC both in vitro and in vivo [25-27]. Here, we confirmed two of our pre- vious observations [28,31] and those of others [32-35]. First, we confirmed that p27-/-mice are larger than p27 +/+ mice, whereas heterozygous p27+/- mice have an intermediate body weight (see Table 2). The increase in body weight in p27-/- mice was due to generalized orga- nomegaly with disproportional enlargement of the spleen, thymus, testis, ovaries, pituitary gland (data not shown). Second, we confirmed that old (> 7-12 months) p27-/- and p27+/- mice develop spontaneous hyperpla- sia and adenomas in the intermediate pituitary lobe, intestine, kidney and thymus that lead to the animals’ death. The data generated in this study indicates that under normal physiological conditions, PEC prolifera- tion in two-month-old mice is similar in all three p27 genotypes (Table 1). However, when the values of prolif- erating cells were compared in 9- to12-month-old mice, proliferating cells were much higher in p27-/- and p27+/- than in p27+/+ mice, suggesting that p27 absence or insufficiency promotes PEC proliferation in the aging animals where hyperplastic and pre-malignant lesions occur (Table 5). This data supports previous stu- dies from Cardon-Cardo’s group [7], which found higher PEC proliferation (Ki-67 labeling) in p27 (-/-) mice as compared to p27+/+ mice. However, they did not provide information whether p27 heterozygous and mice develop PIN and whether the frequency of PIN could be regulated by hormone stimulation or cancer prevention agents. Thus, it appears that the loss of p27Kip1 keeps a high base level of PEC proliferation in old animals that leads to development of hyperplastic, pre-malignant and malignant lesions. When PEC prolif- eration was stimulated by testosterone, a much higher proliferative response was observed in p27+/- and p27-/- than in p27+/+ mice, indicating that p27Kip1 lack or haplo-insufficiency increases the sensitivity of PEC to hormone stimulation. This information is important, because there are sufficient clinical data showing that an increased testosterone circulation level in men is