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Myopodin is an actin-binding protein that shuttles between the nucleus and the cytoplasm. After identifying an enriched CpG island encompassing the transcription site of myopodin, we aimed at evaluating the potential relevance of myopodin methylation in bladder cancer. The epigenetic silencing of myopodin by hypermethylation was tested in bladder cancer cells (n=12) before and after azacytidine treatment. Myopodin hypermethylation was associated with gene expression, being increased in vitro by this demethylating agent. The methylation status of myopodin promoter was then evaluated by methylation-specific polymerase chain reaction (MS-PCR) analyses. Myopodin was revealed to be frequently methylated in a large series of 466 bladder tumours (68.7%). Myopodin methylation was significantly associated with tumour stage (p<0.0005) and tumour grade (p=0.037). Myopodin expression patterns were analysed by immunohistochemistry on tissue arrays containing bladder tumours for which myopodin methylation was assessed (n=177). The presence of low nuclear myopodin expression alone (p = 0.031) or combined with myopodin methylation (p=0.008) was associated with poor survival. Moreover, myopodin methylation in 164 urinary specimens distinguished patients with bladder cancer from controls with a sensitivity of 65.0%, a specificity of 79.8%, and a global accuracy of 75.3%. Thus, myopodin was identified to be epigenetically modified in bladder cancer. The association of myopodin methylation and nuclear expression patterns with cancer progression and clinical outcome, together with its ability to detect bladder cancer patients using urinary specimens, suggests the utility of incorporating myopodin methylation assessment in the clinical management of patients affected by uroepithelial neoplasias.

Testicular specimens from normal men and men with cryptorchidism (CR) or Klinefelter's syndrome (KS) were taken, processed for light microscopy, and stained with the avidin-biotin peroxidase complex method for immunohistochemical detection of testosterone. The Leydig cells were classified by their morphology (normal, multivacuolated, and pleomorphic Leydig cells) and by their staining affinity for anti-testosterone antibodies (T-, T+, and T++ cells), and the average numbers of each cell type for each group of testes were calculated. Normal testes showed morphologically normal interstitial Leydig cells (96.0 +/- 10 per cent) and multivacuolated Leydig cells (4.0 +/- 1 per cent). Cryptorchid testes showed normal Leydig cells (85.8 +/- 11 per cent) and multivacuolated Leydig cells (14.2 +/- 2.3 per cent). Men with KS showed normal Leydig cells (78.9 +/- 9.1 per cent), multivacuolated Leydig cells (9.2 +/- 1.2 per cent), and pleomorphic Leydig cells (11.0 +/- 1.8 per cent). The percentage of T++ cells was higher in normal testes (29.4 +/- 2.1 per cent) than in CR (11.4 +/- 2.2 per cent) and KS testes (6.3 +/- 0.7 per cent). This suggests reduced functional Leydig cell activity in CR and KS. Multivacuolated Leydig cells showed weaker immunostaining than did normal Leydig cells in all the testicular groups. No immunostaining was shown by pleomorphic Leydig cells. Intratubular Leydig cells were only found in CR and KS. Immunostaining was weaker in intratubular Leydig cells than in interstitial Leydig cells. This suggests that intratubular location reduces functional activity of Leydig cells.