Figure 2

Epigenetic inactivation of S100 genes by promoter hypermethylation in medulloblastoma cell lines. (A) Expression analysis of S100 genes in medulloblastoma cell lines. RT–PCR analysis of S100A2, S100A4, S100A6 and S100A10 is shown for cell lines grown in the presence (+) or absence (−) of the demethylating agent 5-aza CdR (5 μ M, 72 h). Probe signal intensities generated for each gene in independent microarray experiments (see Figure 1A) are also given for comparison. RASSF1A (positive methylation-regulated gene) and ACTB (housekeeping) controls are also shown. (B) Representative examples of bisulphite sequence analysis. Two electropherograms showing analysis of three CpG sites within the S100A10 promoter from a highly methylated cell line (D384) and an unmethylated cell line (DAOY). Individual CpG sites are underlined. (C) Representative examples of COBRA analysis of S100A4. PCR products from bisulphite-treated cell line DNA were digested with HpyCH4IV, which cuts the product twice when CpG sites are methylated, generating products of 100 bp (shown) and 26 and 16 bp (not shown). Unmethylated control (U) was normal blood DNA, methylated control (M) was universal methylated DNA (Serologicals Corporation, Livingston, UK). (D) Methylation status analysis of CpG residues within the promoter-associated CpG islands of S100A2, S100A6 and S100A10 and first intron of S100A4 in nine medulloblastoma cell lines, determined by bisulphite sequencing and estimation of relative peak heights. S100A4 PCR products representative of different digestion patterns were sequenced and used to assign methylation levels to samples showing equivalent digestion patterns. Filled circles, >75% methylation; half-filled circles, 25–75% methylation; empty circles, <25% methylation; NA, not analysed.