Taylor et al. BMC Cancer 2010, 10:541 http://www.biomedcentral.com/1471-2407/10/541 numbers of tumors were determined by ANOVA fol- lowed by unpaired Student’s T-test. SAS software was used for statistical analysis. Differences with p < 0.05 were considered significant. Results p27Kip1 deficiency increases prostate epithelial cell proliferation To determine whether the decrease of p27Kip1 in both p27 heterozygous and p27 deficient mice may affect PEC pro- liferation, two-month-old p27+/+, p27+/- and p27-/- mice were injected ip with BrdU two hrs prior to sacrifice and proliferating cells identified by a corresponding anti-BrdU antibody. DLP was predominantly examined, because most PIN and carcinomas in human prostate develop in tissue areas relevant to DLP of mice. As shown in Table 1, the percentage of BrdU-labeled cells in DLP of mice with the above p27 genotypes is very low (< 0.5%), lacking any significant difference in the values between them. 9cRA at 30 mg/kg body weight given for 7 days by gavage did not significantly affect proliferation of normal PEC. Since tes- tosterone plays a significant role in the development and progression of human prostate cancer, we determined whether p27Kip1 deficiency may affect testosterone- induced cell proliferation. Two-month-old p27+/+, p27+/- and p27-/- mice were treated for 7 days with pla- cebo, testosterone, or a combination of testosterone + 9cRA, and BrdU-labeled cells were determined (Table 1). In testosterone-treated animals, BrdU-labeled cells increased in all three p27 genotypes; the most significant increases were in p27+/- and p27-/- mice, from 0.3 ± 0.2% (placebo) to 1.5 ± 0.6% (p < 0.02) in the former and from 0.4 ± 0.2% (placebo) to 1.7 ± 0.3% (p < 0.001) in the latter. The values of BrdU-labeled cells in testosterone-treated animals were significantly higher in p27+/- and p27-/- mice than in p27+/+ mice. This data indicates that either p27 haplo-insufficiency or p27 deficiency can promote tes- tosterone-induced cell proliferation. In animals treated with testosterone + 9cRA, the values of BrdU-labeled cells were close to those of animals treated with testosterone only and there was no significant difference between them for all three p27 genotypes. However, proliferating cells in p27+/- and p27-/- treated with testosterone + 9cRA were higher than those in p27+/+ mice (Table 1), further indi- cating that p27 deficiency can promote proliferation of prostate epithelial cells. p27Kip1 deficiency promotes prostate carcinogenesis To assess the role of p27 deficiency on prostate carcino- genesis, p27+/+, p27+/- and p27-/- mice at the age of two months were sc injected with testosterone, as described previously, to stimulate cell proliferation, and six days latter MNU was administered ip to initiate the neoplastic process. Four days after MNU administration, the animals Page 4 of 12 were randomized in control (MNU-treated) and 9cRA- treated (MNU+9cRA) groups. The latter animals were treated with 9cRA as long as they remained alive. Most p27-/- mice died at the age of 7 to 12 months primarily from pituitary and other tumors: intestinal, kidney, thymus. Therefore, the incidence and frequency of PIN and tumors in all three genotypes were examined and compared within this time frame. Male p27-/- mice at the end of the study were larger in size as compared to p27+/+ mice, whereas p27 +/- mice had intermediate body weight (Table 2). The weight of DLP also increased in p27+/- and further increased in p27-/- mice as compared to p27+/+ mice: from 48.3 ± 13.1 mg in p27 +/+ to 67.2 ± 14.5 mg in p27+/- to 97.6 ± 21.0 mg (p < 0.01) in p27-/-. MNU alone induced PIN in all three p27 genotypes (Table 3). PIN was detected the most in DLP (Fig. 1A-B), where intraductal (Fig. 1C) and invasive (Figs. 1D-arrows) carcinomas were also identified. The incidence and multiplicity of PIN pro- gressively increased, respectively, from 11% and 0.16 ± 0.5 in p27+/+ to 60% and 1.5 ± 0.5 (p < 0.05) in p27+/-, and further to 70% and 2.0 ± 0.4 in p27-/- (p < 0.005) mice (Table 3). Multiplicity of PIN in p27-/- mice was also higher as compared to that in p27+/- mice (p < 0.01). In addition to PIN in DLP, MNU also induced tumors in dis- tant organs (DO): (pituitary, thyroid, lymph node, intestine) of both p27+/- and p27-/- mice. 9cRA suppresses prostate carcinogenesis irrespective of p27Kip1 expression Treatment of animals with 9cRA for 7-12 months reduced by 10-15% the body weight of all three p27 genotypes, with lack of significant difference between control, placebo-treated and 9cRA-treated animals (Table 2). 9cRA did not significantly decrease DLP weight in p27+/+ and p27+/- mice, but reduced DLP weight in p27-/- mice, from 97.6 ± 21.0 mg in placebo to 80.5 ± 18.6 mg (p < 0.02) in 9cRA-treated mice (Table 2). 9cRA reduced PIN in all three p27 genotypes. Thus, in p27+/+ mice PIN decreased from 11% for inci- dence and 0.16 ± 0.5 for frequency in placebo-treated to zero in 9cRA-treated animals. In these animals no pros- tate or distant tumors were detected (Table 3). 9cRA was also efficacious in both p27+/- and p27-/- mice. In p27+/- mice, 9cRA decreased PIN from 1.5 ± 0.4 (pla- cebo) to 1.1 ± 0.6 (p < 0.05), and in p27-/- mice from 2.0 ± 0.4 (placebo) to 1.4 ± 0.4 (p < 0.005). 9cRA appar- ently was not efficacious and did not reduce the number of tumors in distant organs (Table 3). 9cRA was unable to suppress hormone-induced prostate carcinogenesis We also determined whether testosterone + estradiol hormone stimulation might promote MNU-induced car- cinogenesis in p27 deficient mice and affect the