Cheryl L. Paul

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

Glutathione-S-Transferases (GSTs) comprise a family of isoenzymes that provide protection to mammalian cells against electrophilic metabolites of carcinogens and reactive oxygen species. Previous studies have shown that the CpG-rich promoter region of the π-class gene GSTP1 is methylated at single restriction sites in the majority of prostate cancers. In order to understand the nature of abnormal methylation of the GSTP1 gene in prostate cancer we undertook a detailed analysis of methylation at 131 CpG sites spanning the promoter and body of the gene. Our results show that DNA methylation is not confined to specific CpG sites in the promoter region of the GSTP1 gene but is extensive throughout the CpG island in prostate cancer cells. Furthermore we found that both alleles are abnormally methylated in this region. In normal prostate tissue, the entire CpG island was unmethylated, but extensive methylation was found outside the island in the body of the gene. Loss of GSTP1 expression correlated with DNA methylation of the CpG island in both prostate cancer cell lines and cancer tissues whereas methylation outside the CpG island in normal prostate tissue appeared to have no effect on gene expression.

A distinct sequence (ATAAA)n separates methylated and unmethylated domains at the 5'-end of the GSTP1 CpG island.

What defines the boundaries between methylated and unmethylated domains in the genome is unclear. In this study we used bisulfite genomic sequencing to map the boundaries of methylation that flank the 5′- and 3′-ends of the CpG island spanning the promoter region of the glutathione S-transferase (GSTP1) gene. We show that GSTP1 is expressed in a wide range of tissues including brain, lung, skeletal muscle, spleen, pancreas, bone marrow, prostate, heart, and blood and that this expression is associated with the CpG island being unmethylated. In these normal tissues a marked boundary was found to separate the methylated and unmethylated regions of the gene at the 5′-flank of the CpG island, and this boundary correlated with an (ATAAA)19–24 repeated sequence. In contrast, the 3′-end of the CpG island was not marked by a sharp transition in methylation but by a gradual change in methylation density over about 500 base pairs. In normal tissue the sequences on either side of the 5′-boundary appear to lie in separate domains in which CpG methylation is independently controlled. These separate methylation domains are lost in all prostate cancer whereGSTP1 expression is silenced and methylation extends throughout the island and spans across both the 5′- and 3′-boundary regions.

Methylated Glutathione S-transferase 1 (mGSTP1) is a potential plasma free DNA epigenetic marker of prognosis and response to chemotherapy in castrate-resistant prostate cancer.

Background:Glutathione S-transferase 1 (GSTP1) inactivation is associated with CpG island promoter hypermethylation in the majority of prostate cancers (PCs). This study assessed whether the level of circulating methylated GSTP1 (mGSTP1) in plasma DNA is associated with chemotherapy response and overall survival (OS).Methods:Plasma samples were collected prospectively from a Phase I exploratory cohort of 75 men with castrate-resistant PC (CRPC) and a Phase II independent validation cohort (n=51). mGSTP1 levels in free DNA were measured using a sensitive methylation-specific PCR assay.Results:The Phase I cohort identified that detectable baseline mGSTP1 DNA was associated with poorer OS (HR, 4.2 95% CI 2.1–8.2; P<0.0001). A decrease in mGSTP1 DNA levels after cycle 1 was associated with a PSA response (P=0.008). In the Phase II cohort, baseline mGSTP1 DNA was a stronger predictor of OS than PSA change after 3 months (P=0.02). Undetectable plasma mGSTP1 after one cycle of chemotherapy was associated with PSA response (P=0.007).Conclusions:We identified plasma mGSTP1 DNA as a potential prognostic marker in men with CRPC as well as a potential surrogate therapeutic efficacy marker for chemotherapy and corroborated these findings in an independent Phase II cohort. Prospective Phase III assessment of mGSTP1 levels in plasma DNA is now warranted.

Localisation of satellite DNA sequences on human metaphase chromosomes using bromodeoxyuridine-labelled probes.

Human highly repeated satellite sequences, cloned into M13, were used as templates to prepare single-stranded DNA probes containing bromodeoxyuridine (BrdUrd) in place of thymine. The probes were hybridised to human metaphase chromosomes and visualised using an indirect immunological detection procedure. The sensitivity and accuracy of the technique were tested using a BrdUrd-labelled probe of known copy number and location: a segment from the 2.5 kb Y chromosome repeat. The procedure proved to be reliable and fast, with a sensitivity similar to that of other in situ hybridisation techniques. The technique was then used to determine the chromosomal locations of a 100 bp repeat from human satellite 3. The satellite 3 probe hybridised to a large number of chromosomes and, surprisingly, the intensity of label at all locations remained unchanged when the slides were washed at a higher stringency. The resolution of the technique was very high and allowed accurate localisation of the satellite sequence. Hybridisation was observed in two regions of the subcentromeric heterochromatin of chromosome 9, in two locations at the centromere and short arm of all the acrocentric autosomes, and at the centromere and long arm of the Y chromosome. In addition the probe hybridised to centromeric heterochromatin in chromosomes 1, 16, 17 and 20. We believe that single-stranded BrdUrd-labelled probes should be very useful for detecting RNA transcripts in cells, and discuss ways by which the procedure could be modified to locate single copy DNA on chromosomes.