Taylor et al. BMC Cancer 2010, 10:541 http://www.biomedcentral.com/1471-2407/10/541 but was unable to suppress MNU+hormone-stimulated carcinogenesis. Even in p27+/- mice, a tendency for an increase of PIN by 9cRA is obvious, although the differ- ences in values with placebo-treated animals were not significant. In all three p27 genotypes, the values of PIN incidence are close in placebo- and 9cRA-treated ani- mals. This data supports the results from clinical trials where patients with an increased risk of prostate cancer have been treated with beta carotene, retinol or other antioxidants [36,37]. No efficacy on prostate cancer reduction has been found, but an increase in lung can- cer incidence has been observed that led to preliminary termination of a clinical trial. One potential hypothesis is that 9cRA or its metabolites are not efficacious in highly proliferating cell systems, like PEC stimulated by the above hormones. Our data from short-term experi- ments where mice have been treated with testosterone + 9cRA supports this hypothesis. Another alternative is that in long-term experiments 9cRA may protect PEC from cell death after expiration of testosterone + estra- diol stimulation (60-day releasing pellets). However, in the long-term carcinogenesis study, 9cRA suppressed the incidence and frequency of PIN in MNU-treated mice, indicating that a lack of or decreased p27Kip1 level does not affect the efficacy of 9cRA in suppressing pros- tate carcinogenesis. 9cRA is a ligand of both RARs a,b,g and RXRs a,b,g, suggesting that it may affect various cellular functions by receptor dependent mechanisms [22,25,38]. By ICH we found that RARa and RXRa, principal targets of retinoids and rexinoids, respectively, are expressed in PEC of all three p27 genotypes, sug- gesting a potential receptor-dependent mechanism of 9cRA-induced inhibition of PEC proliferation and carci- nogenesis. An attempt was made to compare RARa and RXRa positive cells in control- and 9cRA-treated ani- mals in a carcinogenesis study (Table 3). However, no difference in the percentage of receptor positive cells was found (data not presented), suggesting receptor independent mechanisms of 9cRA-induced inhibition of prostate carcinogenesis [39]. It has been shown that reti- noids, including 9cRA, at pharmacological doses prefer- entially suppress cell proliferation and may also induce cell differentiation [40]. Some previous studies have also shown that retinoids may induce apoptosis as well, depending on the tissue type and the dose used [9,15]. Our data obtained from the carcinogenesis study indi- cates that 9cRA suppresses cell proliferation but is unable to induce apoptosis. Interestingly, 9cRA did not significantly affect cell proliferation in non-PIN cells, which have low proliferative activity, but significantly reduced proliferating cells in PIN of both p27+/- and p27-/- mice, indicating that 9cRA may have the poten- tial to suppress preferentially pre-malignant stages of prostate carcinogenesis in men independent of the levels Page 10 of 12 of p27Kip1 expression. Surprisingly, we found that in addition to inhibition of cell proliferation, 9cRA can also induce CS in both PIN and non-PIN associated PEC (Table 5 and Fig. 2E-H). In a previous study on mam- mary carcinogenesis, we compared the effect of 9cRA and 4-HPR on CS in MNU-induced mammary tumors and found that 9cRA has a superior role [24]. Data from human prostate also suggest that senescent cells are fre- quently detected in BPH and rarely in prostate cancer, suggesting that malignant transformation of prostate epithelial cells is associated with loss of the potential to senesce [8,41]. Conclusion Our data indicate that p27Kip1 deficiency in PEC pro- motes cell proliferation in an age-dependent manner and increases cellular response to hormone stimulation. p27Kip1 haplo-insufficiency and deficiency stimulate MNU-induced prostate carcinogenesis, suggesting that PIN in human prostate lacking partial or total p27Kip1 expression may have a higher potential to progress and develop malignant phenotype than lesions with wild type p27. The efficacy of 9cRA in suppressing PIN is apparently not p27-dependent, indicating that in poten- tial clinical trials, 9cRA may affect pre-malignant and tumor cells that differentially expressed p27Kip1. The induction of CS by 9cRA in PIN suggests that this bio- marker could be used as a potential biomarker of response in clinical trials for the prevention and treat- ment of prostate cancer. Acknowledgements This study was supported by CA095712 and CN-35112 from NIH. We are thankful to Dr. Vernon Steele, Ph.D., MPH, from NIH for supporting this study and to Dr. Clinton Grubbs from University of Alabama, Birmingham, AL, for providing the 9cRA mixed in the diet. The authors also thank Scott Kennedy for his editorial work. Author details 1University of Illinois at Chicago, Department of Surgery, Division of Surgical Oncology, 840 S. Wood St, Chicago, IL 60612 USA. 2Laboratory of Molecular Toxicology, National Institute of Environmental Health Sciences, Research Triangle Park, NC 27709, USA. 3Department of Endocrinology, Medical University, ul. Zdrave No.2, Sofia, Bulgaria. 4Department of Molecular Pharmacology and Biological Chemistry, Robert H. Lurie Comprehensive Cancer Center, Feinberg School of Medicine, Northwestern University, 303 E. Superior, Lurie 3-113 Chicago, IL 60611 USA. Authors’ contributions WT ran animal experiments and performed p27 gene characterization. AM was involved in animal experiments, p27 gene analysis and hormone stimulation of prostate cells. AA was involved in identification of p27 in prostate cells. HK was involved in the initiating phase of this study and conducted animal studies and p27 gene analysis. NS performed immunocytochemistry and evaluated proliferating cells. AS performed most of the immunocytochemistry for identification of cell proliferation, apoptosis, and cellular senescence. AG was involved in animal experiments and development of p27+/+, p27+/- and p27-/- mice. HK provided p27+/- and p27-/- mice and helped with identification of their genotype. KC was PI of the study and planned, organized, and supervised animal experiments,