Absence of PIWIL2 (HILI) expression in human bladder cancer cell lines and tissues.

BACKGROUND: PIWIL2, a member of Argonaute family of proteins, is exclusively expressed in testis and functions in development and maintenance of germline stem cells. Recently, ectopic expression of PIWIL2 has been reported in a variety of human and mouse tumors. To investigate a potential involvement of PIWIL2 in human bladder cancer, we examined its expression in several human bladder cancer cell lines, normal uroepithelial cell cultures, and some bladder tissues. METHODS: Relative expression of PIWIL2 was determined by real-time quantitative RT-PCR in fifteen bladder carcinoma cell lines, six normal uroepithelial cell cultures and seventy tissue specimens of tumor, tumor margins and morphologically normal tissues of bladder. Specific primers for PIWIL2, TBP and GAPDH (as two internal controls) were used for reverse transcription polymerase chain reaction technique. RESULTS: Real-time qRT-PCR demonstrated high PIWIL2 expression in testis tissue, but at least 240-fold lower expression in all examined cell lines. The highest expression outside testis was observed in one of six primary cultures of normal uroepithelial cells, but even lower expression of PIWIL2 was detected in any of the examined tumor and non-tumor tissues. CONCLUSION: Lack of PIWIL2 expression in most tissues along with its aberrant expression in some tumors candidate the gene as an attractive tumor marker for some neoplasms. However, our study indicates that PIWIL2 does not play a role in carcinogenesis of human bladder carcinoma.

Relationship of SNCG, S100A4, S100A9 and LCN2 gene expression and DNA methylation in bladder cancer.

Microarray analysis of paired cultures of normal and cancerous urothelial cells revealed differences in cytokeratin and adhesion gene expression. Normal cells expressed autocrine growth factor genes more strongly whereas carcinoma cells were distinguished by concomitant expression of urothelial and epidermal differentiation markers. Expression of SNCG, S100A9 and LCN2 was also enhanced. In other cancers, overexpression of SNCG, LCN2 and S100A4 has been ascribed to DNA hypomethylation. We therefore investigated expression and methylation of SNCG, S100A4, S100A9 and LCN2 in urothelial cancer cell lines and tissues. SNCG and S100A4 were overexpressed in some cancer tissues and cell lines, but downregulated in others, whereas LCN2 and S100A9 were upregulated in few cancer cell lines, but regularly in tissues. Normal and cancerous urothelial cells expressing SNCG lacked promoter methylation. SNCG downregulation was associated with hypermethylation and could be reversed by the DNA methyltransferase inhibitor 5-aza-2'-deoxycytidine. S100A4 methylation at regulatory intronic sites and in the promoter region was lowest in leukocytes and fibroblasts, and denser in urothelial cells. Gene expression responded to 5-aza-2'-deoxycytidine. LCN2 promoter methylation was variable and even less consistently related to expression. The S100A9 promoter was partially methylated in nonexpressing cells, but 5-aza-2'-deoxycytidine had no effect. Our data indicate that SNCG methylation is cell type-specific and the gene is hypermethylated in some urothelial cancers. S100A4, S100A9 and LCN2 are genes with moderate CpG-density that show a less stringent relationship between DNA methylation and gene expression. Therefore, changes in methylation of these genes in cancer should be interpreted cautiously.