Aberrant CpG methylation changes occurring during tumour progression include the loss (hypomethylation) and gain (hypermethylation) of methyl groups. control. We have therefore demonstrated the ability of this technique, the identification of CGI exhibiting altered methylation patterns (ICEAMP), to isolate tumour-specific methylated GC-rich sequences. This will allow a comprehensive identification of methylation changes during tumourigenesis and will lead to a better understanding of the processes involved. INTRODUCTION The aberrant methylation of CpG dinucleotides has been widely reported during tumourigenesis in a variety of cancers (for review see 1). The alterations identified and their consequences include the loss of methyl groups, which is thought to buy 931409-24-4 increase chromosomal instability (2). Alternatively, the gain of methyl groups, particularly in CpG islands [CGIs: GC-rich regions of the genome, ~1 kb in length, originally characterised due to their lack of methylation (3)], is linked to the transcriptional repression of the associated genes (for review see 4). Whether such aberrant methylation patterns are a part of the causative process or a result of secondary effects is unclear. However, once established, aberrant methylation patterns are clonally inherited with high fidelity in all progeny cells. The initial methylation change and subsequent alterations buy 931409-24-4 will therefore be represented in the later stages of tumourigenesis. An extensive examination of the differences in methylation (both gains and losses) at different stages of cancer development will lead to a clearer understanding of the mechanisms involved and provide an additional buy 931409-24-4 means of tumour classification. In addition, the identification of aberrantly methylated CGIs will allow identification of those genes disrupted during tumour progression. Finally, identification of the earliest changes in methylation will provide useful biomarkers in cancer diagnosis and treatment. Methods that are currently available for the investigation of methylation changes include restriction landmark genome scanning (5) and genome scanning analysis (6). The method described here sought to identify DNA methylation changes that occur during tumour progression in breast cancers, without prior knowledge of the sequences involved or availability of restriction enzyme recognition sites. We focused specifically on single copy GC-rich regions, such as CGIs, altered by aberrant methylation. Genomic fragments were first isolated from the remainder of the genome by utilizing a methyl-CpG binding domain (MBD) column (7). This comprises the MBD of rat MeCP2 covalently linked to a histidine tag (HMBD), then attached to a Ni-agarose matrix. At Rabbit polyclonal to KLF8 low salt concentration the bulk of the genome will bind to the column; however, under conditions of increasing salt concentration only densely methylated sequences will remain bound, allowing their isolation (7). Previous work with an MBD column has revealed that in addition to low and single copy methylated sequences this tightly bound fraction also contained repetitive elements (8), for example SINEs and LINEs (short and long interspersed nuclear elements). In order to isolate only those CGIs with altered methylation patterns, a process of subtractive hybridisation was employed (using DNA extracted from tumour as tester and from matched normal tissue as driver) to remove these repetitive sequences. Using this method, CGIs and other low copy number GC-rich regions with altered methylation patterns between normal and tumour could be extracted, cloned and then analysed. MATERIALS AND METHODS The methyl-binding domain column The MBD column was constructed and operated following a previously described protocol (7). The Fast Pressure Liquid Chromatography HR 10/2 column (Amersham Pharmacia Biotech) used in this study contained ~8 ml of slurry with ~16 mg/ml of bound HMBD. DNA derived from human female blood (100 g) was digested with 200 U buy 931409-24-4 of ((7). As this technique was used to generate libraries of unmethylated CGIs, this may indicate a methylation change in this region. A number of inserts (10%) have no homology to sequences currently represented in the database. Many of these sequences required several sequencing attempts and contain inserts that are both very GC rich and have a high CpGO/E.. Their sequences may not yet be included in the database. We extended the methylation analysis.