Cytidine methylation of regulatory sequences near the pi-class glutathione S-transferase gene accompanies human prostatic carcinogenesis.

Abstract

Hypermethylation of regulatory sequences at the locus of the a%-el glutathione S-trnsferase gene GSTPI was detected in 20 of 20 human prostatic carcinoma tissue specimens studied but not in normal tissues or prostatic tissues exhibiting benign hyperplasia. In addition, a striking decrease in GSTPI expression was found to accompany human prostatic MATERIALS AND METHODS Immunohistochemical Staiing for GSTP1. Formalin-fixed paraffin-embedded prostatic tissue sections were stained with anti-GSTP1 antiserum (1:100 dilution; Oncor) using an immunoperoxidase method (Vectastain ABC Kit; Vector Laboratories), with either 3,3'-diaminobenzidine (DAB) or 3-amino-9-ethylcarbazole (AEC) as the peroxidase substrate. As a control for the specificity of staining, tissue sections prepared from normal human liver and normal human kidney were also stained with the anti-GSTP1 antiserum. Each ofthe tissues exhibited the characteristic staining pattern previously described (14).Immunoblot Analysis for GSTP1 Polypeptides. Salinewashed cultured prostatic carcinoma cells (15-19) were collected by centrifugation at 14,000 x g for 10 min at room temperature and then lysed in a protein extraction buffer [2% SDS/101% (vol/vol) glycerol/10 mM dithiothreitol in 62 mM Tris'HCl, pH 7.8] by heating to 950C for 10 min. The DNA content of each cell extract was estimated by using a diphenylamine assay (20). Equivalent extracts from each of the cultured cell lines were subjected to immunoblot analysis for GSTP1 and DNA topoisomerase I polypeptides, using specific antisera (rabbit anti-GSTP1 from Oncor; human antitopoisomerase I provided by W. C. Earnshaw, Johns Hopkins University) as previously described (21).Northern Blot Analyss for GSTPI mRNA. Total RNAs were extracted from cultured cells by the method of Chomczynski and Sacchi (22) and then quantitated by using an orcinol assay (23). Purified RNAs (20 ug) were electrophoresed on 1.5% agarose gels in the presence of2.2 M formaldehyde, transferred to Zeta-Probe (Bio-Rad) filters, and then assessed for GSTPI and TOP] mRNA levels by hybridization with specific 32P-labeled cDNA probes (prepared using the Random Primers DNA Labeling System, GIBCO/BRL). Following hybridization at 50CC for 16 hr (in 50%o formamide/7% SDS/0.5% nonfat dry milk/heat-denatured salmon sperm DNA at 200 pg/ml/300 mM NaCl/2 mM EDTA/20 mM sodium phosphate, pH 7.4), blots were washed with 0.3x SSC (lx SSC is 150mM NaCl/15 mM sodium citrate, pH 7.0) and 0.3% SDS for 60 mi at 650C before being exposed to X-Omat AR (Kodak) film at -700C. Plasmid pGSTii1 containing the entire GSTPI coding sequence (24) was obtained from the American Type Culture Collection; plasmid phTOPl-D1 containing 704 bp of human TOP) cDNA (25) was provided by

Detailed methylation analysis of the glutathione S-transferase π (GSTP1) gene in prostate cancer

“…An association between increased GSTP1-1 expression and resistance to cytotoxic drugs has also been established with breast cancer cells (Batist et al, 1986). Conversely, immunohistochemical staining demonstrated a loss of GSTP1-1 expression in 88 of 91 cancer specimens (Lee et al, 1994). The LNCaP prostate cancer cell line that lacks GSTP1-1 protein was shown not to express GSTP1mRNA and to be methylated at speci®c restriction sites in the 5'¯anking region of the gene.…”

“…The prostate cancer cell lines we tested included PC3 and Du145 that express GSTP1-1 and LNCaP that does not express GSTP1-1 (Lee et al, 1994). Following bisulphite treatment of the DNA the core promoter region (PCR1) was ampli®ed (Figure 1a and 2).…”

“…Following bisulphite treatment of the DNA the core promoter region (PCR1) was ampli®ed (Figure 1a and 2). This region contains the NotI and SacII sites, that were shown to correlate with the silencing of the GSTP1 gene in prostate cell lines when methylated (Lee et al, 1994). Direct sequencing of the PCR1 product ( Figure 3a) followed by Genescan analysis (Figure 3b) was used to quantitate the methylation status.…”

“…The LNCaP prostate cancer cell line that lacks GSTP1-1 protein was shown not to express GSTP1mRNA and to be methylated at speci®c restriction sites in the 5'¯anking region of the gene. In addition, in all 20 prostate carcinoma samples that were examined, methylation at a speci®c restriction site (BssHII) was observed, while matched normal prostate DNA was unmethylated (Lee et al, 1994). Methylation analysis was carried out using methylation sensitive restriction endonucleases and consequently was limited by the number of CpG sites in the promoter region that could be assayed.…”

Prostate cancer risk from occupational exposure to polycyclic aromatic hydrocarbons interacting with the GSTP1 Ile105Val polymorphism

“…Phase II enzymes (e.g., glutathione S-transferases (GSTs) and N-acetyltransferases) mediate the conjugation of watersoluble moieties, such as glutathione, which are responsible for detoxification of these reactive metabolites [17]. GSTP1 is involved in the inactivation of cigarette smoke carcinogens, such as BPDE, and other toxic constituents, such as acrolein [18], and GSTP1 is expressed in normal prostate cells [19]. An A to G transition at nucleotide 313 in exon 5 of the GSTP1 gene, which replaces isoleucine (Ile) at codon 105 with valine (Val) within the active site of the enzyme, is associated with reduced enzymatic activity for certain substrates and altered thermostability [20,21].…”