Gene expression and epigenetic discovery screen reveal methylation of SFRP2 in prostate cancer

Aberrant activation of Wnts is common in human cancers, including prostate. Hypermethylation associated transcriptional silencing of Wnt antagonist genes SFRPs (Secreted Frizzled-Related Proteins) is a frequent oncogenic event. The significance of this is not known in prostate cancer. The objectives of our study were to (i) profile Wnt signaling related gene expression and (ii) investigate methylation of Wnt antagonist genes in prostate cancer. Using TaqMan Low Density Arrays, we identified 15 Wnt signaling related genes with significantly altered expression in prostate cancer; the majority of which were upregulated in tumors. Notably, histologically benign tissue from men with prostate cancer appeared more similar to tumor (r = 0.76) than to benign prostatic hyperplasia (BPH; r = 0.57, p < 0.001). Overall, the expression profile was highly similar between tumors of high (≥ 7) and low (≤ 6) Gleason scores. Pharmacological demethylation of PC-3 cells with 5-Aza-CdR reactivated 39 genes (≥ 2-fold); 40% of which inhibit Wnt signaling. Methylation frequencies in prostate cancer were 10% (2/20) (SFRP1), 64.86% (48/74) (SFRP2), 0% (0/20) (SFRP4) and 60% (12/20) (SFRP5). SFRP2 methylation was detected at significantly lower frequencies in high-grade prostatic intraepithelial neoplasia (HGPIN; 30%, (6/20), p = 0.0096), tumor adjacent benign areas (8.82%, (7/69), p < 0.0001) and BPH (11.43% (4/35), p < 0.0001). The quantitative level of SFRP2 methylation (normalized index of methylation) was also significantly higher in tumors (116) than in the other samples (HGPIN = 7.45, HB = 0.47, and BPH = 0.12). We show that SFRP2 hypermethylation is a common event in prostate cancer. SFRP2 methylation in combination with other epigenetic markers may be a useful biomarker of prostate cancer.

IGFBP7 promoter methylation and gene expression analysis in prostate cancer

PURPOSE: IGFBP7 belongs to a family of insulin-like growth factor-1 regulatory binding proteins. IGFBP7 hypermethylation is associated with its down-regulation in various carcinomas. In prostate cancer IGFBP7 down-regulation has been widely reported but to our knowledge the mechanisms behind this event are unknown. We performed a denaturing high performance liquid chromatography screening and validation strategy to profile the methylation status of IGFBP7 in prostate cancer.

MATERIALS AND METHODS: We combined denaturing high performance liquid chromatography and bisulfite sequencing to examine IGFBP7 methylation in a panel of prostate cancer cell lines. Quantitative methylation specific polymerase chain reaction was used to determine methylation levels in prostate tissue specimens of primary prostate cancer, histologically benign prostate adjacent to tumor, high grade prostatic intraepithelial neoplasia and benign prostatic hyperplasia. IGFBP7 gene expression was measured by quantitative methylation specific polymerase chain reaction in cell lines and tissue specimens.

RESULTS: IGFBP7 was methylated in the 4 prostate cancer cell lines DU145, LNCaP, PC-3 and 22RV1. Quantitative methylation specific polymerase chain reaction analysis revealed that promoter methylation was associated with decreased IGFBP7 expression. Quantitative methylation specific polymerase chain reaction showed that IGFBP7 methylation was more frequently detected in prostate cancer (60% (31/52)) and high grade prostatic intraepithelial neoplasia (40% (6/15)) samples compared to histologically benign prostate adjacent to tumor (10%) and benign prostatic hyperplasia (0%) samples.

CONCLUSIONS: To our knowledge this is the first report of aberrant IGFBP7 promoter hypermethylation and concurrent IGFBP7 gene silencing in prostate cancer cell lines. Results demonstrate that CpG methylation of IGFBP7 may represent a novel biomarker of prostate cancer and pre-invasive neoplasms. Thus, future examination of IGFBP7 methylation and expression in a larger patient cohort, including bodily fluids, is justified to further evaluate its role in a diagnostic and prognostic setting.

In silico analysis and DHPLC screening strategy identifies novel apoptotic gene targets of aberrant promoter hypermethylation in prostate cancer

BACKGROUND: Aberrant DNA methylation has been implicated as a key survival mechanism in cancer, whereby promoter hypermethylation silences genes essential for many cellular processes including apoptosis. Limited data is available on the methylation profile of apoptotic genes in prostate cancer (CaP). The aim of this study was to profile methylation of apoptotic-related genes in CaP using denaturing high performance liquid chromatography (DHPLC).

METHODS: Based on an in silico selection process, 13 genes were screened for methylation in CaP cell lines using DHPLC. Quantitative methylation specific PCR was employed to determine methylation levels in prostate tissue specimens (n = 135), representing tumor, histologically benign prostate, high-grade prostatic intraepithelial neoplasia and benign prostatic hyperplasia. Gene expression was measured by QRT-PCR in cell lines and tissue specimens.

RESULTS: The promoters of BIK, BNIP3, cFLIP, TMS1, DCR1, DCR2, and CDKN2A appeared fully or partially methylated in a number of malignant cell lines. This is the first report of aberrant methylation of BIK, BNIP3, and cFLIP in CaP. Quantitative methylation analysis in prostate tissues identified 5 genes (BNIP3, CDKN2A, DCR1, DCR2 and TMS1) which were frequently methylated in tumors but were unmethylated in 100% of benign tissues. Furthermore, 69% of tumors were methylated in at least one of the five-gene panel. In the case of all genes, except BNIP3, promoter hypermethylation was associated with concurrent downregulation of gene expression.

CONCLUSION: Future examination of this "CaP apoptotic methylation signature" in a larger cohort of patients is justified to further evaluate its value as a diagnostic and prognostic marker.

Targeting of photooxidative damage on single-stranded DNA representing the bcr-abl chimeric gene using oligonucleotide-conjugates containing [Ru(phen)3](2+)-like photosensitiser groups

Photooxidative damage was induced predominantly at a single guanine base in a target DNA by irradiation (lambda > 330 nm) in the presence of complementary oligodeoxynucleotide conjugates (ODN-5'-linker-[Ru(phen)3]2+) (phen = 1,10-phenanthroline). The target DNA represents the b2a2 variant of the chimeric bcr-abl gene implicated in the pathogenesis of chronic myeloid leukaemia, and the sequence of the 17mer ODN component of the conjugate (3' G G T A G T T A T T C C T T C T T 5') was complementary to the junction region of the sense strand sequence of this oncogene. Two different conjugates were prepared, both of them by reaction of the appropriate succinimide ester with 5'-hexylamino-derivatised 17mer ODN. In Ru-ODN-1 (7) the linker was -(CH2)6-NHCO-bpyMe (-bpyMe = 4'-[4-methyl-2,2'-bipyridyl]), whereas in Ru-ODN-2 (13) it was -(CH2)6-NHCO-(CH2)3-CONH-phen. Photoexcitation of either of the conjugates when hybridised with the 32P-5'-end-labelled target 34mer 5'T G A C C A T C A A T A A G G A A G A A G21 C C C T T C A G C G G C C 3' (ODN binding site underlined) led to an alkali-labile site predominantly (> 90%) at the G21 base, which is at the junction of double-stranded and single-stranded regions of the hybrid. Greater yields were found with Ru-ODN-1 (7) than with Ru ODN-2 (13). In contrast to this specific cleavage with Ru-ODN-1 (7) or Ru-ODN-2 (13), alkali-labile sites were generated at all guanines when the 34mer was photolysed in the presence of the free sensitiser [Ru(phen)3]2+. Since [Ru(phen)3]2+ was shown to react with 2'-deoxyguanosine to form the diastereomers of a spiroiminodihydantoin derivative (the product from 1O2 reaction), 1O2 might also be an oxidizing species in the case of Ru-ODN-1 (7) and Ru-ODN-2 (13). Therefore to determine the range of reaction, a series of 'variant' targets was prepared, in which G21 was replaced with a cytosine and a guanine substituted for a base further towards the 3'-end (e.g. Variant 3; 5'T G A C C A T C A A T A A G G A A G A A C C G23 C T T C A G C G G32 C C3'). While it was noted that efficient reaction took place at distances apparently remote from the photosensitiser (e.g at G32, but not G23 for Variant 3), this effect could be attributed to hairpinning of the single-stranded region of the target. These results are therefore consistent with the photooxidative damage being induced by a reaction close to the photosensitiser rather than by a diffusible species such as 1O2.

Characterization of a reference material for BCR-ABL (M-BCR) mRNA quantitation by real-time amplification assays:towards new standards for gene expression measurements

Monitoring of BCR-ABL transcripts has become established practice in the management of chronic myeloid leukemia. However, nucleic acid amplification techniques are prone to variations which limit the reliability of real-time quantitative PCR (RQ-PCR) for clinical decision making, highlighting the need for standardization of assays and reporting of minimal residual disease (MRD) data. We evaluated a lyophilized preparation of a leukemic cell line (K562) as a potential quality control reagent. This was found to be relatively stable, yielding comparable respective levels of ABL, GUS and BCR-ABL transcripts as determined by RQ-PCR before and after accelerated degradation experiments as well as following 5 years storage at -20 degrees C. Vials of freeze-dried cells were sent at ambient temperature to 22 laboratories on four continents, with RQ-PCR analyses detecting BCR-ABL transcripts at levels comparable to those observed in primary patient samples. Our results suggest that freeze-dried cells can be used as quality control reagents with a range of analytical instrumentations and could enable the development of urgently needed international standards simulating clinically relevant levels of MRD.

Characterization of protein C receptor expression in monocytes

Many sequelae associated with endotoxaemic-induced shock result from excessive production of the cytokine mediators, tumour necrosis factor alpha (TNF-alpha), interleukin 1 (IL-1) and IL-6 from lipopolysaccharide (LPS)-activated monocytes. Protein C (PC)/activated protein C (APC) has potent cytokine-modifying properties and is protective in animal models and human clinical trials of sepsis. The precise mechanism by which this anti-inflammatory response is achieved remains unknown; however, the recently described endothelial protein C receptor (EPCR) appears to be essential for this function. The pivotal role that monocytes play in the pathophysiology of septic shock led us to investigate the possible expression of a protein C receptor on the monocyte membrane. We used similarity algorithms to screen human sequence databases for paralogues of the EPCR but found none. However, using reverse transcription-polymerase chain reaction (RT-PCR), we detected an mRNA transcribed in primary human monocytes and THP1 cells that was identical to human EPCR mRNA. We also used immunocytochemical analysis to demonstrate the expression of a protein C receptor on the surface of monocytes encoded by the same gene as EPCR. These results confirm a new member of the protein C pathway involving primary monocytes. Further characterization will be necessary to compare and contrast its biological properties with those of EPCR.

Severe aplastic anaemia in association with a unique constitutional translocation 46,XY,t(6;10)(q13;q22)c

Severe aplastic anaemia (SAA) is an uncommon disorder which may be associated with several congenital syndromes. However, it has rarely been described in association with a constitutional karyotypic abnormality. The breakpoint of the balanced t(6:10)(q13:q22) translocation described here does not disrupt any currently recognized gene of haemopoietic or stromal importance. This report also highlights the problems inherent in the use of bone marrow transplantation (BMT) for treating multiply transfused aplastic anaemia patients.

Adenovirus 12 E1A gene detection by polymerase chain reaction in both the normal and coeliac duodenum

A 12 amino acid sequence from the adenovirus 12 E1B protein is homologous at the protein level with a similar 12-mer derived from the wheat protein A-gliadin. It has been suggested that exposure to Ad 12 could sensitise individuals to gliadins with resultant gluten sensitive enteropathy. In this study, the polymerase chain reaction (PCR) was used to analyse duodenal biopsy tissue from patients with coeliac disease for the presence of Ad 12. The sensitivity of the assay system was at least 1 in 10(5) cells and specificity was confirmed both by probing with an internal oligonucleotide and by direct sequencing. Ad 12 sequences were detected in three of 17 patients with adult coeliac disease and in five of 16 adult controls with normal duodenal biopsies. Since exposure to the virus would be predicted to occur in infancy we also studied patients with childhood coeliac disease diagnosed at less than 1 year of age. Ad 12 was positive in three of 10 childhood coeliac patients and one of seven controls. In addition, we studied a cohort of patients who presented with a diarrhoeal illness and associated anti alpha gliadin antibodies in 1983. These patients had duodenal biopsies performed at this time. One of three patients with abnormal histology had detectable Ad 12 while two of 14 with normal findings were positive for Ad 12. Finally, the potential oncogenic nature of Ad 12 prompted examination of a group of patients with intestinal tumours. Ad 12 DNA was, however, in only two of 19 tumour samples tested. These data indicate that Ad 12 can be successfully detected using PCR on paraffin embedded tissue. Furthermore, Ad 12 was detected at a relatively high level in normal duodenum. The results do not, however, support the hypothesis that prior exposure to Ad 12 is implicated in the pathogenesis of coeliac disease.

Autosomal dominant retinitis pigmentosa (ADRP):localization of an ADRP gene to the long arm of chromosome 3

Members of a large pedigree of Irish origin presenting with early onset Type I autosomal dominant retinitis pigmentosa (ADRP) have been typed for D3S47 (C17), a polymorphic marker from the long arm of chromosome 3. Significant, tight linkage of ADRP to D3S47, with a lod score of 14.7 maximizing at 0.00 recombination, has been obtained, hence localizing the ADRP gene (RP1) segregating in this pedigree to 3q.

Integrative analysis of chemo-transcriptomic profiles for drug-feature specific gene expression signatures

One of the major challenges in systems biology is to understand the complex responses of a biological system to external perturbations or internal signalling depending on its biological conditions. Genome-wide transcriptomic profiling of cellular systems under various chemical perturbations allows the manifestation of certain features of the chemicals through their transcriptomic expression profiles. The insights obtained may help to establish the connections between human diseases, associated genes and therapeutic drugs. The main objective of this study was to systematically analyse cellular gene expression data under various drug treatments to elucidate drug-feature specific transcriptomic signatures. We first extracted drug-related information (drug features) from the collected textual description of DrugBank entries using text-mining techniques. A novel statistical method employing orthogonal least square learning was proposed to obtain drug-feature-specific signatures by integrating gene expression with DrugBank data. To obtain robust signatures from noisy input datasets, a stringent ensemble approach was applied with the combination of three techniques: resampling, leave-one-out cross validation, and aggregation. The validation experiments showed that the proposed method has the capacity of extracting biologically meaningful drug-feature-specific gene expression signatures. It was also shown that most of signature genes are connected with common hub genes by regulatory network analysis. The common hub genes were further shown to be related to general drug metabolism by Gene Ontology analysis. Each set of genes has relatively few interactions with other sets, indicating the modular nature of each signature and its drug-feature-specificity. Based on Gene Ontology analysis, we also found that each set of drug feature (DF)-specific genes were indeed enriched in biological processes related to the drug feature. The results of these experiments demonstrated the pot- ntial of the method for predicting certain features of new drugs using their transcriptomic profiles, providing a useful methodological framework and a valuable resource for drug development and characterization.

ChIPping away at gene regulation

The coordinated regulation of gene expression in higher eukaryotes is complex and poorly understood. Recent technological advances have allowed the first insights into these networks on a genome-wide scale. These investigations have identified transcription factor target sites in the genome and successfully predicted cooperative interactions with other factors. However, a detailed understanding of the processes that coordinate gene expression remains elusive. Here, we highlight the advances that have been made using current methods, and the need for new technologies to address the gaps in our knowledge and to map these complex pathways further.

Genome-wide association study for sight-threatening diabetic retinopathy reveals association with genetic variation near the GRB2 gene

AIMS/HYPOTHESIS: Diabetic retinopathy is a serious complication of diabetes mellitus and can lead to blindness. A genetic component, in addition to traditional risk factors, has been well described although strong genetic factors have not yet been identified. Here, we aimed to identify novel genetic risk factors for sight-threatening diabetic retinopathy using a genome-wide association study.

METHODS: Retinopathy was assessed in white Australians with type 2 diabetes mellitus. Genome-wide association analysis was conducted for comparison of cases of sight-threatening diabetic retinopathy (n = 336) with diabetic controls with no retinopathy (n = 508). Top ranking single nucleotide polymorphisms were typed in a type 2 diabetes replication cohort, a type 1 diabetes cohort and an Indian type 2 cohort. A mouse model of proliferative retinopathy was used to assess differential expression of the nearby candidate gene GRB2 by immunohistochemistry and quantitative western blot.

RESULTS: The top ranked variant was rs3805931 with p = 2.66 × 10(-7), but no association was found in the replication cohort. Only rs9896052 (p = 6.55 × 10(-5)) was associated with sight-threatening diabetic retinopathy in both the type 2 (p = 0.035) and the type 1 (p = 0.041) replication cohorts, as well as in the Indian cohort (p = 0.016). The study-wide meta-analysis reached genome-wide significance (p = 4.15 × 10(-8)). The GRB2 gene is located downstream of this variant and a mouse model of retinopathy showed increased GRB2 expression in the retina.

CONCLUSIONS/INTERPRETATION: Genetic variation near GRB2 on chromosome 17q25.1 is associated with sight-threatening diabetic retinopathy. Several genes in this region are promising candidates and in particular GRB2 is upregulated during retinal stress and neovascularisation.