Hypermethylation Trigger of the Glutathione-S-Transferase Gene (GSTP1) in Prostate Cancer Cells
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Oncogene (2002) 21, 1048 ± 1061 ã 2002 Nature Publishing Group All rights reserved 0950 ± 9232/02 $25.00 www.nature.com/onc Hypermethylation trigger of the glutathione-S-transferase gene (GSTP1) in prostate cancer cells Jenny Z Song1, Clare Stirzaker1, Janet Harrison1, John R Melki1 and Susan J Clark*,1,2,3 1Kanematsu Laboratories, Sydney Cancer Centre, Royal Prince Alfred Hospital, Camperdown, NSW 2050, Australia; 2Faculty of Medicine, University of Sydney, Sydney, NSW, 2006, Australia; 3CSIRO, Molecular Science, Sydney Laboratory, PO Box 184, North Ryde, NSW 1670, Australia Understanding what triggers hypermethylation of tumour CpG islands, span promoter regions of house-keeping suppressor genes in cancer cells is critical if we are to genes and are typically unmethylated. The biological discern the role of methylation in the oncogenic process. signi®cance of DNA methylation in vertebrates is still CpG sites in CpG island promoters, that span most tumour debated, but it is thought to play a major role in gene suppressor genes, remain unmethylated in the normal cell, silencing of repeated sequences (Turker and Bestor, despite the fact that CpG sites are the prime target for de 1997) and possibly plays a role in modulating tissue- novo methylation by the DNA methyltransferases. The speci®c gene expression (Warnecke and Clark, 1999). CpG island-associated with the GSTP1 gene is an What is clear is that DNA methylation is critical for intriguing example of a CpG rich region which is normal development, as embryos die at implantation in susceptible to hypermethylation in the majority of prostate knockout mice that are lacking the DNA methyltrans- tumours and yet is unmethylated in the normal prostate ferase gene Dnmt1 (Li et al., 1992). Three additional cell. In this study we evaluate a number of factors DNA methyltransferases have now been described; purported to be involved in hypermethylation to test their Dnmt2 displays no activity in vitro, but Dnmt 3a and 3b role in triggering hypermethylation of GSTP1 in prostate have been shown to exhibit de novo methylation activities cancer DU145 and LNCaP cells. We ®nd that hyper- (Hsieh, 1999) and are also critical for embryo develop- methylation is not associated with (1) elevated expression ment (Okano et al., 1999). of the DNA methyltranferases, or (2) removal of Sp1 In human cancers the DNA methylation pattern of transcription factor binding sites in the CpG island or (3) the genome is altered, with the repeated region of the removal of CpG island boundary elements or (4) prior gene genome often demethylated and the CpG island- silencing. Instead our results support a model that requires associated genes often hypermethylated. In particular a combination of prior gene silencing and random `seeds' of the CpG islands spanning the promoter regions of methylation to trigger hypermethylation of the GSTP1 tumour-suppressor genes are methylated early in cancer gene in the prostate cancer cell. We propose that the progression (Baylin et al., 2001). Multiple tumour GSTP1 gene is initially silenced in the prostate cancer and suppressor or tumour-associated genes are methylated random sites of methylation accumulate that result in in any one cancer cell but the sub-set of genes that are subsequent hypermethylation and chromatin remodelling. susceptible to methylation are cancer-subtype speci®c Oncogene (2002) 21, 1048 ± 1061. DOI:10.1038/sj/onc/ (Melki et al., 1999). Methylation of the CpG island 1205153 region of genes is associated with their inactivation and therefore abnormal DNA methylation has been Keywords: DNA methylation; CpG islands; bisulphite described as a contributory factor equal in importance genomic sequencing; GSTP1; prostate cancer 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 Introduction changes in the cancer cell and in particular what triggers the hypermethylation of normally unmethy- Methylation at the 5'-position of cytosine in CpG lated gene promoter regions is a critical but unan- dinucleotides is a common modi®cation of DNA in swered question in cancer biology. vertebrate genomes. However not all CpG sites are One means to address the mechanism that is susceptible to methylation resulting in compartmentali- responsible for aberrant hypermethylation in cancer is zation of the `methyl' genome. Repeated sequence DNA to determine what protects CpG islands normally from and the CpG-depleted regions of the genome are mostly methylation. Interestingly the CpG dinucleotide is the hypermethylated, whereas the CpG rich clusters termed 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 *Correspondence: SJ Clark; E-mail: [email protected] Received 27 August 2001; revised 25 October 2001; accepted 31 changes (Warnecke and Clark, 1998). A number of October 2001 possibilities have been proposed. Firstly, that the DNA Hypermethylation trigger in prostate cancer JZ Song et al 1049 Figure 1 GSTPI vector contructs. (A) Map of the GSTPI gene showing the position of the 7 exons, and the ATAAA repeat element. Below is an expanded map of the GSTP1 shuttle vector map containing the 2.3 kb XbaI/SnaBI cloned into pBluescriptSK+. The restriction enzyme sites in the GSTP1 and plasmid sequence and co-ordinates are shown. (B) CpG map of the region of the GSTP1 gene from CpG 756 to CpG +10 is shown. The scale is marked relative to co-ordinates from GenBank Accession Number M24485). The modi®cations to the various GSTP1 constructs are indicated in each of the CpG maps. *, marks the location of HpaII sites within the GSTP1 CpG island map methyltransferase enzymes DNMT1, 3a and 3b are Over expression of DNMT1 can result in aberrant moderately elevated in cancer cells (Robertson et al., promoter CpG island methylation in vitro (Vertino et 1999) and this may be sucient to elicit the changes al., 1996), however a bi-allelic knockout of the same gene observed in the cancer cell genomic methylation pattern. in a cancer cell did not abolish maintenance of the CpG Oncogene Hypermethylation trigger in prostate cancer JZ Song et al 1050 island methylation (Rhee et al., 2000), suggesting other al., 1998; Cookson et al., 1997; Esteller, 2000; Lee et DNA methyltransferases may be responsible. Secondly, al., 1994; Millar et al., 1999; Moskaluk et al., 1997). protein factors may protect CpG islands from methyla- We previously reported that the methylation pattern tion by binding interference. Mutation of Sp1 sites in the across the GSTP1 CpG island in the prostate cancer adenine phosphoribosyl transferase (APRT) gene led to cell line LNCaP is extensive and the gene is silent the hypermethylation of the associated CpG island in (Millar et al., 1999, 2000). Essentially all the CpG sites transfection assays and transgenic mouse studies are methylated extending past the ATAAA repeat (Brandeis et al., 1994; Macleod et al., 1994). Thirdly, motif through the promoter region and into the body RNA transcripts from CpG islands may themselves of the gene (Figure 2). In contrast, the GSTP1 gene protect the islands either directly or indirectly in that is actively expressed in the prostate cancer cell line combination with 5MeC-DNA glycosylase (Jost et al., DU145, shows a methylation pro®le more typical of 1997). Finally, replication timing or chromatin structure normal prostate cells where the ATAAA repeat may inhibit CpG island methylation (Kass et al., 1997). sequence separates the upstream methylated CpG sites The CpG island promoter region spanning the from the unmethylated CpG island. Previously, by glutathione S-transferase (GSTP1) gene becomes methy- direct bisulphite PCR sequencing, we showed that the lated in the majority of prostate tumours. The gene is promoter region of the GSTP1 gene is completely expressed and unmethylated in all normal tissues and is unmethylated (Millar et al., 1999). However by cloning not commonly methylated in other cancers, with the PCR product and sequencing individual clones we exception of liver and breast cancer (Esteller et al., ®nd that about one-third of the molecules in the 1998; Jhaveri and Morrow, 1998; Tchou et al., 2000). DU145 cells show a small amount of methylation Intriguingly no mutations or deletions have been around the start of transcription (Figure 2). This reported for GSTP1 in prostate cancer, however the pattern of low level methylation around the transcrip- gene is inactivated and both alleles are commonly tion start site is similar to the methylation pattern in methylated (Millar et al., 1999). Therefore, the GSTP1 normal liver in which expression of GSTP1 is reduced CpG island becomes an ideal candidate to study what (Millar et al., 2000). triggers its hypermethylation in prostate cancer cells. We have previously described the methylation pro®le across DNA methyltransferase expression in LNCaP versus the CpG island of the GSTP1 gene in normal and DU145 prostate cancer cells and found that the gene is extensively methylated and inactivated in prostate cancer To determine if the dierence in the methylation state cells, including the prostate cancer cell line LNCaP of the endogenous GSTP1 gene in LNCaP and (Millar et al., 1999, 2000). In contrast, the cancer cell line DU145 is related simply to the level of the DNA DU145 behaves more like a normal prostate cell with methyltransferases between the two cell lines we GSTP1 gene essentially unmethylated and expressed. A determined the expression of the DNA methyltrans- detailed methylation analysis in normal cells revealed a ferase enzymes DNMT1, 2, 3a and 3b by quantitative marked boundary of methylation at the 5' ¯ank of the of real-time RT ± PCR (Taqman). Figure 3 shows the the island that separated the methylated and unmethy- relative expression pro®le of the dierent DNA lated regions of the gene and this boundary correlated methyltransferases.