Hypermethylation trigger of the glutathione-S-transferase gene (GSTP1) in prostate cancer cells Jenny Z Song 1 , Clare Stirzaker 1 , Janet Harrison 1 , John R Melki 1 and Susan J Clark* ,1,2,3 1 Kanematsu Laboratories, Sydney Cancer Centre, Royal Prince Alfred Hospital, Camperdown, NSW 2050, Australia; 2 Faculty of Medicine, University of Sydney, Sydney, NSW, 2006, Australia; 3 CSIRO, Molecular Science, Sydney Laboratory, PO Box 184, North Ryde, NSW 1670, Australia Understanding what triggers hypermethylation of tumour suppressor genes in cancer cells is critical if we are to discern the role of methylation in the oncogenic process. CpG sites in CpG island promoters, that span most tumour suppressor genes, remain unmethylated in the normal cell, despite the fact that CpG sites are the prime target for de novo methylation by the DNA methyltransferases. The CpG island-associated with the GSTP1 gene is an intriguing example of a CpG rich region which is susceptible to hypermethylation in the majority of prostate tumours and yet is unmethylated in the normal prostate cell. In this study we evaluate a number of factors purported to be involved in hypermethylation to test their role in triggering hypermethylation of GSTP1 in prostate cancer DU145 and LNCaP cells. We find that hyper- methylation is not associated with (1) elevated expression of the DNA methyltranferases, or (2) removal of Sp1 transcription factor binding sites in the CpG island or (3) removal of CpG island boundary elements or (4) prior gene silencing. Instead our results support a model that requires a combination of prior gene silencing and random ‘seeds’ of methylation to trigger hypermethylation of the GSTP1 gene in the prostate cancer cell. We propose that the GSTP1 gene is initially silenced in the prostate cancer and random sites of methylation accumulate that result in subsequent hypermethylation and chromatin remodelling. Oncogene (2002) 21, 1048 – 1061. DOI:10.1038/sj/onc/ 1205153 Keywords: DNA methylation; CpG islands; bisulphite genomic sequencing; GSTP1; prostate cancer Introduction Methylation at the 5’-position of cytosine in CpG dinucleotides is a common modification of DNA in vertebrate genomes. However not all CpG sites are susceptible to methylation resulting in compartmentali- zation of the ‘methyl’ genome. Repeated sequence DNA and the CpG-depleted regions of the genome are mostly hypermethylated, whereas the CpG rich clusters termed CpG islands, span promoter regions of house-keeping genes and are typically unmethylated. The biological significance of DNA methylation in vertebrates is still debated, but it is thought to play a major role in gene silencing of repeated sequences (Turker and Bestor, 1997) and possibly plays a role in modulating tissue- specific gene expression (Warnecke and Clark, 1999). What is clear is that DNA methylation is critical for normal development, as embryos die at implantation in knockout mice that are lacking the DNA methyltrans- ferase gene Dnmt1 (Li et al., 1992). Three additional DNA methyltransferases have now been described; Dnmt2 displays no activity in vitro, but Dnmt 3a and 3b have been shown to exhibit de novo methylation activities (Hsieh, 1999) and are also critical for embryo develop- ment (Okano et al., 1999). In human cancers the DNA methylation pattern of the genome is altered, with the repeated region of the genome often demethylated and the CpG island- associated genes often hypermethylated. In particular the CpG islands spanning the promoter regions of tumour-suppressor genes are methylated early in cancer progression (Baylin et al., 2001). Multiple tumour suppressor or tumour-associated genes are methylated in any one cancer cell but the sub-set of genes that are susceptible to methylation are cancer-subtype specific (Melki et al., 1999). Methylation of the CpG island region of genes is associated with their inactivation and therefore abnormal DNA methylation has been described as a contributory factor equal in importance to gene mutation and gene deletion in mediating gene silencing in cancer (Costello and Plass, 2001; Jones and Laird, 1999). What triggers the DNA methylation changes in the cancer cell and in particular what triggers the hypermethylation of normally unmethy- lated gene promoter regions is a critical but unan- swered question in cancer biology. One means to address the mechanism that is responsible for aberrant hypermethylation in cancer is to determine what protects CpG islands normally from methylation. Interestingly the CpG dinucleotide is the prime target for methylation by the DNA methyltrans- ferase enzymes, however CpG sites in CpG islands are never methylated during development when the rest of the genome is undergoing major de novo methylation changes (Warnecke and Clark, 1998). A number of possibilities have been proposed. Firstly, that the DNA Oncogene (2002) 21, 1048 – 1061 ª 2002 Nature Publishing Group All rights reserved 0950 – 9232/02 $25.00 www.nature.com/onc *Correspondence: SJ Clark; E-mail: susan.clark@molsci.csiro.au Received 27 August 2001; revised 25 October 2001; accepted 31 October 2001