Figure 2

Methylation pattern of TMS1/ASC. Panel A – graphical representation of the structure of TMS1/ASC gene showing position of the three exons. Panel B shows the location of the CpG dinucleotides. Panel C shows the postion of the primers used for MS-PCR and bisulfite sequencing in the promoter region of TMS1/ASC. Positions are indicated relative to translation start site. Panel D shows MS-PCR analysis of TMS1/ASC gene on the different prostate cancer cell lines. LNCaP exhibits complete methylation of TMS1/ASC gene. PC3 and Du145 have both the methylated as well as the unmethylated allele and are consequently expressed. NC-negative control, U-unmethylated allele, M-methylated allele. The methylation pattern correlates with the bisulfite genomic sequencing shown on panel E. Completely methylated cytosines (black arrow) in LNCaP are not converted to thymidine following bisulfite treatment and show up on the C lane whereas partially methylated cytosines in the CpG's are seen both in the T lane as well as in the C lane in PC3 and Du145 cell lines. Positions are indicated relative of the translation start site. Panel F shows representative examples of 5 prostate cancer tissue samples analyzed by MS-PCR and gel electrophoresis. Presence of a band in lanes marked as UM indicates presence of unmethylated allele and a band in the lanes marked M denotes a methylated allele; NC-negative control; PC-positive control.