Supplementary Methods, Figure Legends 1-4 from Global Methylation Pattern of Genes in Androgen-Sensitive and Androgen-Independent Prostate Cancer Cells
ARTICLE ABSTRACT
Promoter DNA methylation of CpG islands is an important epigenetic mechanism in cancer development. We have characterized the promoter methylation profile of 82 genes in three prostate cancer cell lines (LNCaP, PC3, and DU145) and two normal prostate cell lines (RWPE1 and RWPE2). The methylation pattern was analyzed using a Panomics gene array system that consists of immobilized probes of known gene promoters on a nitrocellulose membrane. Methylation binding protein–purified methylated DNA was hybridized on the membrane and detected by the chemiluminescence method. We analyzed methylation profile in normal (RWPE1) versus cancerous cells and androgen receptor (AR)–sensitive (LNCaP) versus AR-negative cells (DU145 and PC3). Our study shows that >50% of the genes were hypermethylated in prostate cancer cells compared with 13% in normal cell lines. Among these were the tumor suppressor (RB, TMS1, DAPK, RBL1, PAX6, and FHIT), cell cycle (p27KIP1 and CDKN2A), transporters (MDR1, MLC1, and IGRP), and transcription factor (STAT1, CIITA, MYOD, and NPAT) genes. Relative methylation pattern shows that most of these genes were methylated from 5-fold to >10-fold compared with the normal prostate cells. In addition, promoter methylation was detected for the first time in target genes such as RIOK3, STAT5, CASP8, SRBC, GAGE1, and NPAT. A significant difference in methylation pattern was observed between AR-sensitive versus AR-negative cancer cells for the following genes: CASP8, GPC3, CD14, MGMT, IGRP, MDR1, CDKN2A, GATA3, and IFN. In summary, our study identified candidate genes that are methylated in prostate cancer. Mol Cancer Ther; 9(1); 33–45
