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One of the genes induced by demethylation in LNCaP cells was 14-3-3σ. Using MSP conditions described in section 5.2.2 it was confirmed that this gene is completely methylated in LNCaP cells and underwent partial demethylation upon treatment with 5Aza-2’dC resulting in mRNA induction detected by RT-PCR and Northern blot analysis (Figure 17, a and b; data not shown). Furthermore, detection of methylated cytosines with higher resolution by bisulfite sequencing demonstrated a minimal degree of CpG methylation of the 14-3-3σ gene in PrECs, whereas dense methylation was evident in the DNA derived from the LNCaP cell line (Figure 19a).

0 0,2 0,4 0,6 0,8 1 1,2 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 0 0,2 0,4 0,6 0,8 1 1,2 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25

14-3-3σ

LNCaP

PrECs

Du-145 TSU-Pr1 LAPC.4 BPH-1

PrECs M U 14-3-3σ M U M U M U M U M U M U M U

b

c

a

Figure 19 Methylation status and protein expression of 14-3-3σ in PCa cell lines

(a) The pattern of CpG methylation of the 14-3-3σ locus as determined by bisulfite sequencing. Genomic DNA isolated from PrECs and PCa cell line LNCaP was treated with bisulfite and analysed as described in Methods. Depicted is a schematic representation of the CpG distribution around transcription start site of 14-3-3σ. The detailed description of the graphic features is given in the legend for Figure 18. (b) 14-3-3σ -specific MSP analysis of PrECs, PCa and BPH-1 cell lines. Genomic DNA was isolated from exponentially proliferating cells, treated with sodium bisulfite and used as a template for MSP analysis with primers specific for the methylated (M) and unmethylated (U)

14-3-3σ allele. BPH-1: benign prostate hyperplasia cells immortalized with SV40 large T antigen; PrECs: human primary prostate epithelial cells. (c) Detection of 14-3-3σ protein expression. Western blot analysis was performed with extracts from exponentially growing cells using an affinity-purified rabbit 14-3-3σantiserum. Detection of α-tubulin was employed as a loading control.

MSP analysis extended to other cell lines revealed that 14-3-3σ is also methylated in the PCa cell lines PPC1 and LAPC4, but not in a cell line established from a benign prostate hyperplasia (BPH1) or in primary prostate epithelial cells (Figure 19b). By Western blot analysis, an inverse correlation between the degree of CpG methylation and protein expression was identified (Figure 19c): LNCaP cells, which display complete CpG methylation of 14-3-3σ, were devoid of 14-3-3σ

expression; PPC1 cells, which have methylated and unmethylated 14-3-3σ alleles, showed a significant down-regulation of 14-3-3σ protein expression. The cell lines Du-145, PC3 and TSU-Pr1 did not reveal any CpG methylation in the 14-3-3σ gene and showed relatively high levels of 14-3-3σ protein expression. The highest level of 14-3-3σ expression was detected in the PrECs, which lack CpG methylation of the 14-3-3σ gene. Interestingly, the cell lines Du-145 and PC3 harbor p53 mutations, whereas LNCaP cells express wild-type p53. This correlation suggests that silencing of 14-3-3σmay potentially alleviate the requirement to inactivate p53 in PCa.

In order to facilitate detection of aberrant methylation in a larger number of specimens the CpG methylation patterns obtained for other candidate genes were used to assign the positions of MSP-primers (indicated in Figure 18). The respective MSP-primers were tested for their specificity and sensitivity (data not shown). Only for CUTL2 reliable MSP-conditions could not be established. For analysis of genes previously known to be silenced by CpG methylation in other tumor types, the respective published MSP-primers were tested and used for MSP analysis. In total 14 genes (including 14-3-3σ) which displayed significant CpG methylation as determined by bisulfite sequencing or MSP in one of the cell lines PC3, LNCaP or Du-145 were examined by MSP in a panel of five prostate cancer cell lines, BPH1, PrECs and in the bladder carcinoma cell line TSU-Pr1 (Figure 20, left panel).

The analysis of candidates revealed, that the genes initially identified in selected PCa cell lines also displayed CpG methylation in other PCa cell lines and, occasionally in the cell line BPH1. 9 of the 14 analyzed genes did not display CpG methylation in primary prostate epithelial cells (PrECs). Therefore, CpG methylation of these 9 genes is a specific feature of cancerous prostate epithelial cells. APOD, DDB2, GSTM1 and RIS1 displayed partial CpG methylation in PrECs (Figure 20). However, the degree of CpG methylation of DDB2, APOD and GSTM1 appeared to be significantly elevated in most of the PCa-cell lines when compared to normal PrECs (Figure 20), suggesting a PCa-specific increase in CpG methylation of these

Figure 20 Comparative analysis of CpG methylation and gene expression

Left panel: MSP analysis of a series of PCa cell lines and primary prostate epithelial cells (left panel). The MSP-analysis was done using two primers sets (M = methlyated; U = unmethlyated) specific for the indicated genes. BPH1 = benign prostate hyperplasia cells immortalized with SV40 large T antigen. Right panel: RT-PCR analysis of the indicated genes in non-transformed and tumor cells. D1-D4 samples represent primary prostate epithelial cells (PrECs) from four different donors. ß-actin and γ- tubulin (TUBG2) were used as additional standards to EF1α.

genes and potential subsequent silencing. CpG methylation of SFRP1, GPX3, GSTM1 and APOD was also detected in the bladder carcinoma cell line TSU-Pr1. The expression level of 9 genes which showed selective or preferential CpG methylation in PCa was analyzed in order to determine whether CpG methylation correlates with reduced or absent expression of the respective genes. RT-PCR analysis of cDNAs obtained from normal prostate epithelial cells derived from 4 healthy donors and 4 PCa cell lines revealed several distinct patterns of mRNA expression (Figure 20, right panel). The CpG methylation interrogated by the MSP- primers used here largely correlated with reduced gene expression in the case of SFRP1, DKK3, GPX3, COX2, GSTM1, APOD and p57, whereas the detected CpG methylation of DDB2 and HPGD was not accompanied by decreased gene expression. In the case of HPGD the expression was even induced in 3 PCa samples, which clearly showed CpG methylation. These results indicate that CpG methylation of a promoter should not be interpreted as proof for its transcriptional repression.