CD4+CD25+ T regulatory (Treg) cells were initially described for their ability to suppress autoimmune diseases in animal models. An emerging interest is the potential role of Treg cells in cancer development and progression because they have been shown to suppress antitumor immunity. In this study, CD4+CD25− T cells cultured in conditioned medium (CM) derived from tumor cells, RENCA or TRAMP-C2, possess similar characteristics as those of naturally occurring Treg cells, including expression of Foxp3, a crucial transcription factor of Treg cells, production of low levels of IL-2, high levels of IL-10 and TGF-β, and the ability to suppress CD4+CD25− T cell proliferation. Further investigation revealed a critical role of tumor-derived TGF-β in converting CD4+CD25− T cells into Treg cells because a neutralizing Ab against TGF-β, 1D11, completely abrogated the induction of Treg cells. CM from a nontumorigenic cell line, NRP-152, or irradiated tumor cells did not convert CD4+CD25− T cells to Treg cells because they produce low levels of TGF-β in CM. Finally, we observed a reduced tumor burden in animals receiving 1D11. The reduction in tumor burden correlated with a decrease in tumor-derived TGF-β. Treatment of 1D11 also reduced the conversion of CD4+ T cells into Treg cells and subsequent Treg cell-mediated suppression of antitumor immunity. In summary, we have demonstrated that tumor cells directly convert CD4+CD25− T cells to Treg cells through production of high levels of TGF-β, suggesting a possible mechanism through which tumor cells evade the immune system.

The results indicated that estrogen could activate ERK pathway through the non-genomic ERalpha pathway, leading to proliferation of PrSCs.

Prostate cancers often develop insensitivity to TGF-beta to gain a growth advantage. In this study, we explored the status of promoter methylation of TGF-beta receptors (TbetaRs) in a prostate cancer cell line, LNCaP, which is insensitive to TGF-beta. Sensitivity to TGF-beta was restored in cells treated with 5-Aza-2'-deoxycytidine (5-Aza), as indicated by an increase in the expression of phosphorylated Smad-2, type I (TbetaRI), and type II (TbetaRII) TGF-beta receptors, and a reduced rate of proliferation. The same treatment did not significantly affect a benign prostate cell line, RWPE-1, which is sensitive to TGF-beta. Mapping of methylation sites was performed by screening 82 potential CpG methylation sites in the promoter of TbetaRI and 33 sites in TbetaRII using methylation-specific PCR and sequence analysis. There were six methylation sites (-365, -356, -348, -251, -244, -231) in the promoter of TbetaRI. The -244 site was located in an activator protein (AP)-2 box. There were three methylated sites (-140, +27, +32) in the TbetaRII promoter and the -140 site was located in one of the Sp1 boxes. Chromatin immunoprecipitation analysis demonstrated DNA binding activity of AP-2 in the TbetaRI promoter and of Sp1 in the TbetaRII promoter after treatment with 5-Aza. To test whether promoter methylation is present in clinical specimens, we analyzed human prostate specimens that showed negative staining for either TbetaRI or TbetaRII in a tissue microarray system. DNA samples were isolated from the microarray after laser capture microdissection. Methylation-specific PCR was performed for TbetaRI (six sites) and TbetaRII (three sites) promoters as identified in LNCaP cells. A significant number of clinical prostate cancer specimens lacked expression of either TbetaRI and/or TbetaRII, especially those with high Gleason's scores. In those specimens showing a loss of TbetaR expression, a promoter methylation pattern similar to that of LNCaP cells was a frequent event. These results demonstrate that insensitivity to TGF-beta in some prostate cancer cells is due to promoter methylation in TbetaRs.

It is known that human benign prostatic hyperplasia might arise from an estrogen/androgen (E/T) imbalance. We studied the response of castrated rat prostate to different ratios of circulating E/T. The castrated male Wistar rats were randomly injected with E/T at different ratios for 4 weeks. The prostates of E/T (1:100) group showed a distinct prostatic hyperplasia response by prostatic index, hematoxylin and eosin staining, and quantitative immunohistochemical analysis of alpha-smooth muscle actin (SMA). In this group, cells positive for Vimentin, non-muscle myosin heavy chain (NMMHC) and proliferating cell nuclear antigen (PCNA) increased in the stroma and epithelium. Furthermore, the mRNA levels of smooth muscle myosin heavy chain (SMMHC) and NMMHC increased. So E/T at a ratio of 1:100 can induce a stromal hyperplastic response in the prostate of castrated rats. The main change observed was an increase of smooth muscle cells, whereas some epithelial changes were also seen in the rat prostates.

In a preliminary study, we observed that TGF-beta1 induced both proliferation and growth arrest in prostatic stromal cells, depending on the concentration of TGF-beta1 used in the culture medium. In this study, we explored possible mechanisms of this dual effect of TGF-beta. Primary cultures of prostatic stromal cells, established from clinical surgical specimens and treated with low doses of TGF-beta1 (0.001-0.01 ng/ml), resulted in an increase in cell proliferation. The addition of neutralizing antibody against platelet-derived growth factor (PDGF)-BB, but not anti-PDGF-AA, abrogated this stimulatory effect of TGF-beta1. TGF-beta1 treatment resulted in a dose-related increase in PDGF-BB production as measured by ELISA. Cells underwent growth arrest at high concentrations of TGF-beta1 (1.0 and 10 ng/ml). An inhibitor of cyclin-dependent kinase (cdk), p15INK4b, was up-regulated at both transcript and protein levels in these cultures by TGF-beta1 in a dose-related manner as determined by RT-PCR and Western blot analysis. The transcript, but not the protein, for another cdk inhibitor, p21Cip1, was up-regulated with treatment of TGF-beta1 to these cells. Levels of other cdk inhibitors, such as p16INK4a and p27Kip1, were constitutively expressed in prostatic stromal cells and were not significantly affected by TGF-beta1 treatment. Finally, the growth arrest effect of TGF-beta1 was abrogated when antisense oligonucleotides to p15INH4b, but not p21Cip1, were added to the culture medium. These data indicate that the dual effect of TGF-beta1 is mediated, at least, by up-regulation of PDGF-BB and p15INK4b, respectively.