Construction of PYCARD-inducible system in two cancer cell lines, LNCaP (prostate cancer) and DLD1 (colorectal cancer)

Epigenetic gene silencing by aberrant DNA methylation is one of the important mechanisms leading to the loss of key cellular pathways in tumorigenesis. We previously reported that PYCARD (PYD and CARD domain containing), an apoptosis inducing gene, was frequently hypermethylated in prostate cancer in a tumor-specific manner (46/51: 90%), by methylation specific PCR (MSP). In order to examine the relationship between methylation status and level of the PYCARD protein expression in prostate cancer, we further performed immunohistochemical analyses of the 51 MSP-analyzed prostate cancer specimens and observed reduced or absent protein expression in tumors with aberrant methylation status of PYCARD. In contrast, PYCARD was unmethylated and expressed in normal prostatic tissues including epithelial, basal and inflammatory cells. Furthermore, we tried to identify downstream genes of PYCARD by microarray analyses using PYCARD inducible LNCaP prostate cancer cells. Interestingly, PYCARD induction upregulated PYDC1, another PYCARD homolog that associates with PYCARD and accelerates inflammation and apoptosis. Our present results suggest that aberrant methylation of PYCARD is an extremely frequent event and contributes to escape from apoptosis by reducing PYCARD expression in prostate cancer. Two cancer-derived cell lines, LNCaP_PYCARD and DLD1_PYCARD, that allow tetracycline-regulated PYCARD expression were constructed. The RNAs from LNCaP_PYCARD and DLD1_PYCARD with (4 days tetracycline treatment) or without tetracycline were prepared and analyzed by using Agilent whole human genome microarray (4×44K) v2.