A, mRNA levels assessed by quantitative RT-PCR in G1 cells (n = 2) after 1, 3, or 6 h of treatment with 3-adiol (10?6 m). that this may represent an epigenetic event favoring inactivation of the GSTP1 locus by methylation. Moreover, abrogation of ER/eNOS UK-371804 function by 3-adiol emphasizes the significance of circulating or locally produced sex steroid hormones or their metabolites in PCa biology with relevant clinical/therapeutic implications. Prostate cancer (PCa) is the most commonly diagnosed cancer in men in industrialized countries, with the highest incidence in North America (1). PCa, an androgen-dependent tumor, is highly sensitive to perturbation of intratumoral steroid biosynthesis and metabolism of exogenous ligands: androgens but also estrogens and their metabolites. It is now recognized that the combined action, and specifically an imbalance in androgens and estrogens ratio, is critical to PCa development, maintenance, and progression (2, 3). UK-371804 Indeed, a finely tuned balance between estrogens and androgens and the relative expression of the estrogen receptor (ER) subtypes, ER in the stroma and ER in the epithelial compartments of the human prostate (4C8), have been invoked as causative in the etiology of prostate disease (3, 9). The complexity of PCa pathophysiology is enhanced by other signaling molecules such as nitric oxide (NO) and oxygen. We have recently revealed a novel and pivotal function of ER and endothelial NO synthase (eNOS) in the acquisition of an aggressive PCa phenotype (10). Specifically we demonstrated that activation of the ER/eNOS pathway is crucial for tumor progression within the prostate microenvironment, highly sensitive to local changes in hormonal levels and oxygen tension. Estrogens are key signaling molecules regulating various physiological processes, cell growth, development, and differentiation, and also playing a role in many pathological processes in hormone-dependent diseases. Binding of estrogens to ERs, particularly ER in the human UK-371804 prostate epithelium, produces genomic effects (11, 12) that regulate gene transcription. The estrogen-ER complex, once bound to its regulatory site, the estrogen-responsive element (ERE), can interact with adjacent transcription factors and recruit a variety of cofactors, thus inducing modifications of the chromatin resulting in activation or repression of target genes (13C16). A second key molecule, NO, the product of eNOS, is a free radical involved in many biological processes, among which is angiogenesis. Recently it has been shown that activated eNOS can translocate into the nucleus (17C20) where it binds ER (10). Formation of an eNOS/ER combinatorial complex determines localized remodeling of chromatin, leading to transcriptional activation of previously identified prognostic genes (expression of the GST P1-1 protein by TMA in a retrospective cohort of PCa patients characterized by very long follow-up (10). We confirmed the loss of GST P1-1, consistent with data in the literature (24, 25), and correlated it with decreased ANK2 disease-specific survival (DSS; Fig. 1B). Of interest, a limited number of PCa samples (14 of 126) retained GST P1-1 expression (Fig. 1A, and model of cell lines established from PCa patients (10, 26) by documenting a significant decrease of GSTP1 mRNA and protein expression and enzymatic activity, consistent with data in the literature (Fig. 2, ACC, and Supplemental Fig. 1, published on The Endocrine Society’s Journals Online web site at http://mend.endojournals.org) in cells from patients with worse favorable outcome (G1 and G2 cells). As expected, cell lines established from benign prostatic hyperplasia (BPH) showed strong GSTP1 expression and activity. Surprisingly, DNA methylation-sensitive restriction assay (Fig. 2D) revealed that silencing did not involve promoter methylation,.