Douglas Spencer Millar

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.

Genetic and epigenetic instability of human bone marrow mesenchymal stem cells expanded in autologous serum or fetal bovine serum

Culture of mesenchymal stem cells (MSCs) under conditions promoting proliferation and differentiation, while supporting genomic and epigenetic stability, is essential for therapeutic use. We report here the extent of genome-wide DNA gains and losses and of DNA methylation instability on 170 cancer-related promoters in bone marrow (BM) MSCs during culture to late passage in medium containing fetal bovine serum (FBS) or autologous serum (AS). Comparative genomic hybridization indicates that expansion of BMMSCs elicits primarily telomeric deletions in a subpopulation of cells, the extent of which varies between donors. However, late passage cultures in AS consistently display normal DNA copy numbers. Combined bisulfite restriction analysis and bisulfite sequencing show that although DNA methylation states are overall stable in culture, AS exhibits stronger propensity than FBS to maintain unmethylated states. Comparison of DNA methylation in BMMSCs with freshly isolated and cultured adipose stem cells (ASCs) also reveals that most genes unmethylated in both BMMSCs and ASCs in early passage are also unmethylated in uncultured ASCs. We conclude that (i) BMMSCs expanded in AS or FBS may display localized genetic alterations, (ii) AS tends to generate more consistent genomic backgrounds and DNA methylation patterns, and (iii) the unmethylated state of uncultured MSCs is more likely to be maintained in culture than the methylated state.

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.

Methylation sequencing from limiting DNA: embryonic, fixed, and microdissected cells.

It is frequently useful to determine the methylation state of samples containing limited amounts of DNA such as from embryos, or from fixed tissue samples in which DNA is degraded or difficult to isolate. By modification of the standard protocols for DNA preparation and bisulfite treatment, it is possible to obtain DNA methylation sequence data for such samples. We present methods for bisulfite treatment of embryos, fixed sections, and samples obtained by laser capture microdissection, and discuss the additional experimental considerations required when working with small numbers of cells or degraded DNA samples.

Methylation of a CpG island within the promoter region of the KAI1 metastasis suppressor gene is not responsible for down-regulation of KAI1 expression in invasive cancers or cancer cell lines

The molecular basis for downregulation of the KAI1 metastasis suppressor gene in invasive and metastatic human cancers is unknown. We have used bisulphite methylation analysis of DNA from paraffin-embedded invasive bladder tumour samples and from bladder cancer cell lines to determine if hypermethylation of a CpG island within the KAI1 promoter is responsible for this effect. Representative invasive tumour cell lines were also exposed to 5-aza-2-deoxycytidine. We found no evidence for hypermethylation of the CpG island and suggest that mechanisms other than promoter hypermethylation are responsible for reduced KAI1 expression in invasive bladder tumours and tumour cell lines.

Comparison of a novel HPV test with the Hybrid Capture II (hcII) and a reference PCR method shows high specificity and positive predictive value for 13 high-risk human papillomavirus infections.

BACKGROUND It is well established that human papillomavirus (HPV) infection is highly related to the development of precursor lesions of cervical cancer and uterine cancers. However, for a pre-cancerous lesion to develop, a persistent infection with a high-risk type HPV is necessary. The Digene Hybrid Capture II (hcII) assay is the only FDA approved method used in conjunction with cytology for HPV screening of women older than 30. The hcII has moderate sensitivity (64.7%) and is dependent on the cellular content of samples, rendering occasionally false positive and false negative results. OBJECTIVE This study aims to evaluate the performance of a new HPV diagnostic kit (High-Risk HPV detection kit, manufactured by Human Genetic Signatures (HGS), Sydney, Australia). METHODS The method under evaluation was assessed by comparing the results obtained from testing 834 cervical specimens with the HGS method and the Digene hcII method, using genotyping as the reference standard. RESULTS Results of the study showed that the specificity and positive predictive value of the HGS High-Risk HPV detection test are significantly greater than those of the Digene hcII test. Overall the HGS HPV assay provides a more accurate system for the detection of high-risk HPV strains, with simpler technical use compared with PCR-sequencing methods.

Improved detection of gastrointestinal pathogens using generalised sample processing and amplification panels

Summary We aimed to streamline the diagnosis of gastrointestinal disease by producing multiplexed real time polymerase chain reaction (PCR) panels employing universal sample processing for DNA and RNA containing pathogens. A total of 487 stored, previously characterised stool samples comprising bacterial, viral, protozoan and Clostridium difficile positive samples were tested using four multiplexed real time PCR panels. A further 81 pre-selected clinical samples from a teaching hospital were included to provide an independent validation of assay performance. Improved sensitivity was achieved using the protozoan panels and 16 more mixed infections were observed compared to tests with conventional methods. Using the C. difficile panels, 100% sensitivity was achieved when compared to the gold standard of toxigenic culture. In addition, hypervirulent strains including ribotype 027 could be identified directly from primary sample without the need for ribotyping methods. Bacterial and viral panels detecting Salmonella, Shigella, Campylobacter, Yersinia enterocolitica, Listeria monocytogenes, norovirus groups I and II, rotavirus A, astrovirus, sapovirus, rotavirus B, adenovirus and adenovirus 40/41 performed as well as conventional methods, whilst allowing detection in 3 hours from processing to result. Multiplex real time PCR panels with universal sample preparation allow streamlined, rapid diagnosis of gastrointestinal pathogens whilst extending the characterisation of pathogens present in stool samples from affected patients.