Hypermethylation of regulatory sequences at the locus of the a%-el glutathione S-trnsferase gene GSTPI was detected in 20 of 20 human prostatic carcinoma tissue specimens studied but not in normal tissues or prostatic tissues exhibiting benign hyperplasia. In addition, a striking decrease in GSTPI expression was found to accompany human prostatic MATERIALS AND METHODS Immunohistochemical Staiing for GSTP1. Formalin-fixed paraffin-embedded prostatic tissue sections were stained with anti-GSTP1 antiserum (1:100 dilution; Oncor) using an immunoperoxidase method (Vectastain ABC Kit; Vector Laboratories), with either 3,3'-diaminobenzidine (DAB) or 3-amino-9-ethylcarbazole (AEC) as the peroxidase substrate. As a control for the specificity of staining, tissue sections prepared from normal human liver and normal human kidney were also stained with the anti-GSTP1 antiserum. Each ofthe tissues exhibited the characteristic staining pattern previously described (14).Immunoblot Analysis for GSTP1 Polypeptides. Salinewashed cultured prostatic carcinoma cells (15-19) were collected by centrifugation at 14,000 x g for 10 min at room temperature and then lysed in a protein extraction buffer [2% SDS/101% (vol/vol) glycerol/10 mM dithiothreitol in 62 mM Tris'HCl, pH 7.8] by heating to 950C for 10 min. The DNA content of each cell extract was estimated by using a diphenylamine assay (20). Equivalent extracts from each of the cultured cell lines were subjected to immunoblot analysis for GSTP1 and DNA topoisomerase I polypeptides, using specific antisera (rabbit anti-GSTP1 from Oncor; human antitopoisomerase I provided by W. C. Earnshaw, Johns Hopkins University) as previously described (21).Northern Blot Analyss for GSTPI mRNA. Total RNAs were extracted from cultured cells by the method of Chomczynski and Sacchi (22) and then quantitated by using an orcinol assay (23). Purified RNAs (20 ug) were electrophoresed on 1.5% agarose gels in the presence of2.2 M formaldehyde, transferred to Zeta-Probe (Bio-Rad) filters, and then assessed for GSTPI and TOP] mRNA levels by hybridization with specific 32P-labeled cDNA probes (prepared using the Random Primers DNA Labeling System, GIBCO/BRL). Following hybridization at 50CC for 16 hr (in 50%o formamide/7% SDS/0.5% nonfat dry milk/heat-denatured salmon sperm DNA at 200 pg/ml/300 mM NaCl/2 mM EDTA/20 mM sodium phosphate, pH 7.4), blots were washed with 0.3x SSC (lx SSC is 150mM NaCl/15 mM sodium citrate, pH 7.0) and 0.3% SDS for 60 mi at 650C before being exposed to X-Omat AR (Kodak) film at -700C. Plasmid pGSTii1 containing the entire GSTPI coding sequence (24) was obtained from the American Type Culture Collection; plasmid phTOPl-D1 containing 704 bp of human TOP) cDNA (25) was provided by

BackgroundCachexia, also known as muscle wasting, is a complex metabolic condition characterized by loss of skeletal muscle and a decline in physical function. Muscle wasting is associated with cancer, sarcopenia, chronic obstructive pulmonary disease, end-stage renal disease, and other chronic conditions and results in significant morbidity and mortality. GTx-024 (enobosarm) is a nonsteroidal selective androgen receptor modulator (SARM) that has tissue-selective anabolic effects in muscle and bone, while sparing other androgenic tissue related to hair growth in women and prostate effects in men. GTx-024 has demonstrated promising pharmacologic effects in preclinical studies and favorable safety and pharmacokinetic profiles in phase I investigation.MethodsA 12-week double-blind, placebo-controlled phase II clinical trial was conducted to evaluate GTx-024 in 120 healthy elderly men (>60 years of age) and postmenopausal women. The primary endpoint was total lean body mass assessed by dual energy X-ray absorptiometry, and secondary endpoints included physical function, body weight, insulin resistance, and safety.ResultsGTx-024 treatment resulted in dose-dependent increases in total lean body mass that were statistically significant (P < 0.001, 3 mg vs. placebo) and clinically meaningful. There were also significant improvements in physical function (P = 0.013, 3 mg vs. placebo) and insulin resistance (P = 0.013, 3 mg vs. placebo). The incidence of adverse events was similar between treatment groups.ConclusionGTx-024 showed a dose-dependent improvement in total lean body mass and physical function and was well tolerated. GTx-024 may be useful in the prevention and/or treatment of muscle wasting associated with cancer and other chronic diseases.

Urinary continence following an anatomical approach to radical prostatectomy was evaluated in 593 consecutive patients, 547 (92%) of whom achieved complete urinary control. Stress incontinence was present in 46 patients (8%), of whom 34 (6%) wore 1 or fewer pads per day and 2 (0.3%) required placement of an artificial sphincter. No patient was totally incontinent. Age, weight of the prostate, prior transurethral resection of the prostate, pathological stage and preservation or wide excision of the neurovascular bundles had no significant influence on preservation of urinary control. These data suggest that anatomical factors rather than preservation of autonomic innervation may be responsible for the improved urinary control associated with an anatomical approach to radical prostatectomy.

Bone morphogenetic proteins (BMPs) are members of the transforming growth factor-beta superfamily and signal through a number of membrane receptors. We have previously demonstrated that the loss of expression of BMP receptors (BMPRs) type IA, -IB, and -II (BMP-RIA, -RIB, and -RII) correlates with Gleason score in prostate cancer patients. To evaluate the prognostic value of this observation, we used immunohistochemistry to investigate the expression of BMPRs in association with disease progression in 60 patients. The results demonstrated a significant association between the loss of expression of the three BMPRs and Gleason score and clinical stage. However, only the loss of expression of BMP-RII showed a statistically significant association with 5-year survival rate (P<0.05) and biochemical recurrence-free rate following radical prostatectomy (P<0.005). To elucidate the effect of an abnormal BMP signaling in prostate cancer cells, we transfected dominant-negative BMP-RII (BMP-RIIDN) into the human prostate cancer cell line, PC3M. When a stable clone overexpressing BMP-RIIDN was inoculated subcutaneously into nude mice, the tumor growth rate was approximately 10 times that of control and parental cell line. These observations, taken together, indicate that the loss of BMP-RII expression as measured by immunohistochemistry may be a prognostic marker in prostate cancer patients, and that the loss of BMP-RII function may result in increased tumorigenicity in human prostate cancer cells.

Purpose-Androgen deprivation therapy is associated with fracture risk in men with prostate cancer. We assessed the effects of toremifene, a selective estrogen receptor modulator, on fracture incidence in men receiving androgen deprivation therapy during a 2-year period.Materials and Methods-In this double-blind, placebo controlled phase III study 646 men receiving androgen deprivation therapy for prostate cancer were assigned to toremifene (80 mg by mouth daily) and 638 were assigned to placebo. Subjects were followed for 2 years. The primary study end point was new vertebral fractures. Secondary end points included fragility fractures, bone mineral density and lipid changes.Results-The 2-year incidence of new vertebral fractures was 4.9% in the placebo group vs 2.5% in the toremifene group, a significant relative risk reduction of 50% (95% CI -1.5 to 75.0, p = 0.05). Toremifene significantly increased bone mineral density at the lumbar spine, hip and femoral neck vs placebo (p <0.0001 for all comparisons). There was a concomitant decrease in markers of bone turnover (p <0.05 for all comparisons). Toremifene also significantly improved lipid profiles. Venous thromboembolic events occurred more frequently with toremifene than placebo with 7 subjects (1.1%) in the placebo group experiencing a venous thromboembolic event vs 17 (2.6%) in the toremifene group. Other adverse events were similar between the groups.Conclusions-Toremifene significantly decreased the incidence of new vertebral fractures in men receiving androgen deprivation therapy for prostate cancer. It also significantly improved bone mineral density, bone turnover markers and serum lipid profiles. The intended therapeutic effect of ADT is to reduce testosterone to castrate levels. Because estradiol is derived from the peripheral conversion of testosterone by aromatase, ADT also markedly decreases serum estradiol levels, which may result in unintended estrogen deficiency side effects. Estradiol is critical to bone formation and bone resorption in men. 4 The impact of these changes is decreased BMD and increased incidence of clinical fractures. 5,6 Fractures are an important cause of morbidity in men worldwide. After age 50 years 1 in 4 men experience a clinical fracture. 7 A third of hip fractures occur in men and they are more likely than women to die after hip fracture. Side effects of ADT induced estrogen deficiency are not limited to increased fracture risk. ADT increases serum cholesterol and triglycerides 11 by a mechanism that likely involves estrogen receptor mediated changes in hepatic expression of apoprotein genes. 12 Furthermore, the relatively larger reduction in testosterone levels compared with estradiol can be clinically manifested as gynecomastia. 13 Toremifene is a second-generation SERM. 14 In a small prospective study toremifene increased BMD of the hip and spine in men receiving ADT. 15 In this large randomized placebo controlled trial we evaluated whether toremifene decreased the incidence of new vertebral fractures in men...