Taylor et al. BMC Cancer 2010, 10:541 http://www.biomedcentral.com/1471-2407/10/541 (CDKs) selectively bind to cyclin D1- D3, E, A, B, form- ing complexes that are variably expressed during the cell cycle. When inhibited by p27Kip1, p21Wef1/Cip1, or p16Ink4a, CDKs can suppress cell cycle progression by modulating pRb phosphorylation, leading to inhibition of E2F transcription factors and further to suppression of DNA replication [11,12]. p27Kip1 may also cooperate with other cell cycle suppressor genes and thus further inhibit cell proliferation and carcinogenesis [13,14]. For example, 100% of mice deficient in both p27Kip1 and PTEN (phosphatase and tensin homolog deleted from chromosome 10) (PTEN +/-; p27-/-) developed prostate tumors within 4-6 months vs. 50% of those with the PTEN mutation only [15-17]. Recently, Besson et al. [18] discovered an oncogenic activity of p27Kip1 that causes stem cell expansion and a multiple tumor pheno- type. They generated a knock-in mouse in which four amino acid substitutions in the CDKN1b gene product prevented its interaction with cyclins and CDKs (p27CK*) and found tumors in multiple organs, includ- ing: lung, pituitary, retina, adrenals, ovary, spleen, and lymphomas. No data has been published on the effects of p27Kip1 deficiency on chemically-induced prostate carcinogenesis and on the sensitivity of PEC to retinoids. Studies from the Fero et al. [19] group have shown that p27-/- and, to a lesser extent, p27+/- mice are more susceptible than p27+/+ mice to radiation and chemically induced carcinogenesis. They have found that partial reduction in p27 expression in p27+/- mice can also sensitize cells in a tissue specific manner to undergo malignant transformation. However, they did not examine prostate glands (personal communication). In human tumors haplo-insufficiency is not a frequent phenomenon. However, a moderate decrease in protein expression of certain tumor suppressors, including p27Kip1, may also promote the neoplastic process [20]. Most chemopreventive and antitumor agents, including retinoids, affect normal and tumor cells by inhibiting cell proliferation, and this has been associated with increased expression of cell cycle suppressors [21]. It has been sug- gested that retinoids which induce cell differentiation and suppress cell proliferation can up-regulate p27kip1 and thereby inhibit cell cycle progression [22]. Others [23] and we [24] recently observed that 9cRA can sup- press prostate carcinogenesis when given to rats treated with a carcinogen or to transgenic mice that sponta- neously develop prostate tumors [25]. Our previous stu- dies on Noble rats revealed that, among various retinoids, 9cRA but not 4-(Hydroxyphenyl) retinamide (4-HPR) reduced the incidence and multiplicity of prostate intrae- pithelial neoplasia (PIN) [24]. The molecular mechanisms involved in inhibition of prostate carcinogenesis by 9cRA and other retinoids are poorly understood. 9cRA is a ligand of both retinoic acid receptors (RARs a,b,g) and Page 2 of 12 retinoid × receptors (RXRs a,b,g), suggesting receptor dependent mechanisms of cell and tumor growth inhibi- tion [22]. However, retinoids may also affect normal and tumor cells by receptor independent pathways [26,27]. In this study we assessed the role of p27 deficiency on prostate cell proliferation, response to hormonal stimula- tion, and to treatment with retinoids. We also examined whether p27Kip1 deficiency may affect MNU-induced prostate carcinogenesis and the efficacy of 9cRA in sup- pressing the neoplastic process. We found that p27Kip1 deficiency promotes prostate cell proliferation and carci- nogenesis but does not affect the efficacy of 9cRA in sup- pressing PIN and tumor development. Methods Mice p27+/+, p27+/- and p27-/- mice were generated in our lab by crossbreeding p27+/- female with p27-/- male mice. p27 deficient mice (+/- and -/-) were obtained from Kiyokawas’ lab and have been developed based on the c57BL/6 mouse background. Mouse genotype was assessed by PCR using DNA isolated from small tail tis- sue samples when the animals were 14 days old, as described previously [28]. For the long-term carcinogen- esis study, male p27+/+, p27+/- and p27-/- mice were followed for up to 12 months. Most p27-/- mice died from pituitary and intestinal tumors at the age of 7 to 12 months, as has been reported previously [19,28]. The animals were housed at the Biology Research Laboratory at the University of Illinois, Chicago, and their breeding and treatment were conducted according to the require- ments listed in the Guide for the Care and Use of Laboratory Animals issued by the University’s Animal Care Committee. Animals had free access to water and food, Teklad 4% Purina mouse/rat diet (Harland Teklad, Madison, WI). Hormone stimulation of prostate cell proliferation Two separate studies were performed. In the short-term study, two-month-old mice of all three p27 genotypes were treated for 7 days with testosterone (Sigma Chem. Co., St. Louis) mixed in purified sesame oil. Each animal received 3 sc injections of testosterone, 1.0 mg/kg body weight, 2 days apart in a volume of 0.1 ml. In the long- term chemoprevention study, two experiments were per- formed. In the first, two-month-old p27+/+, p27+/- and p27-/- mice were first treated with testosterone, as described above, and then, on the sixth day after initia- tion of hormone stimulation, MNU was injected ip. In the second chemoprevention experiment, mice were first sc implanted with a testosterone pellet (12.5 mg/ pellet: Cat No: SA-151, Innovative Research of America, Saratoga, FL). Six days later the animals were ip injected with MNU to initiate the neoplastic process. Four days