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. 2001 Nov;159(5):1815–1826. doi: 10.1016/S0002-9440(10)63028-3

Figure 3.

Figure 3.

Analysis of GSTP1 expression and of GSTP1 CpG island methylation for prostate cancer (PCA) case 96 and case 419. Both case 96 and case 419 showed GSTP1 hypermethylation affecting only one of two GSTP1 alleles in PCA DNA by the 5-mCpG-sensitive restriction endonuclease/PCR assay (see Figure 2 ▶ and Table 1 ▶ ). Immunohistochemical staining with anti-GSTP1 antibodies revealed an absence of GSTP1 expression in PCA cells (arrowheads) versus normal cells (arrows) in case 96 (A), but an abundance of GSTP1 expression in PCA cells (arrowheads) in case 419 (B). PCA cells in case 419 nonetheless appeared to express prostate-specific antigen (C) and prostate-specific acid phosphatase (D) as evidenced by immunohistochemical staining with appropriate antibodies. E: DNA from case 96 and from case 419 was subjected to analysis using the 5-mC-sensitive restriction endonuclease-PCR assay described for Figure 2 ▶ . DNA from matched normal (normal) and neoplastic (tumor) prostate tissues was left untreated (U; lanes 1, 4, 7, and 10), or was treated with HpaII (H; lanes 2, 5, 8, and 11), which cuts CCGG but not C5-mCGG, or treated with MspI (M; lanes 3, 6, 9, and 12), which cuts CCGG and C5-mCGG, before being subjected to PCR amplification using oligonucleotide primers targeting a polymorphic [ATAAA]n repeat sequence near the GSTP1 regulatory region. DNA from both of the PCA cases was also subjected to bisulfite genomic sequencing analysis (F), using an assay capable of distinguishing CpG dinucleotide methylation patterns at both maternal and paternal GSTP1 alleles (see Materials and Methods). For each case, a minimum of eight PCR clones was sequenced; the fraction of PCR clones with 5-mC at each CpG site is indicated for each polymorphic [ATAAA]n repeat allele using the gray scale provided. For case 96, although the extent of CpG dinucleotide methylation throughout each GSTP1 CpG island allele was different, both GSTP1 alleles displayed CpG dinucleotide hypermethylation, particularly near known cis regulatory elements. For case 419, GSTP1 DNA hypermethylation appeared to be present on only one of two GSTP1 CpG island alleles.