Characterization of Glucocorticoid Receptor Promoter Methylation in Breast Cancer

Epidemiological studies have identified psychological stress as a significant risk factor in breast cancer. The stress response is regulated by the HPA axis in the brain and is mediated by glucocorticoid receptor (GR) signalling. It has been found that early life events can affect epigenetic programming of GR, and methylation of the GR promoter has been reported in colorectal tumourigenesis. Decreased GR expression has also been observed in breast cancer. In addition, it has been previously demonstrated that unliganded GR can serve as a direct activator of the BRCA1 promoter in mammary epithelial cells. We propose a model whereby methylation of the GR promoter in the breast significantly lowers GR expression, resulting in insufficient BRCA1 promoter activation and an increased risk of developing cancer. Antibody-based methylated DNA enrichment was followed by qPCR analysis (MeDIP-qPCR) in a novel assay developed to detect locus-specific methylation levels. It was found that 13% of primary breast tumours were hypermethylated at the GR proximal promoter whereas no methylation was detected in normal tissue. RT-PCR and 5’ RACE analysis identified exon 1B as the predominant alternative first exon in the breast. Tumours methylated near exon 1B had decreased GR expression compared to unmethylated samples, suggesting that this region is important for transcriptional regulation of GR. It was also determined that GR and BRCA1 expression was decreased in breast tumour compared to normal tissue. Furthermore, the relative expression of GR and BRCA1 measured by qRT-PCR was correlated in normal tissue but this association was not found in tumour tissue. From this, it appears that lower GR levels with associated decreased BRCA1 expression in tissues may be a predisposing factor for breast cancer. Based on these results we propose a role for GR as a potential tumour suppressor gene in the breast due to its association with BRCA1, also a tumour suppressor gene, as well as its consistently decreased expression in breast tumours and methylation of its proximal promoter in a subset of cancer patients.

Methylation status of oestrogen receptor-a-gene promoter sequences in human ovarian epithelial cell lines.

We have determined the methylation status of the CpG island of the oestrogen receptor gene in seven human ovarian cell lines. Cell lines expressing oestrogen receptor showed no evidence of hypermethylation. In three of four cell lines that produced no detectable oestrogen receptor protein, hypermethylation was observed at the NotI site of the CpG island. These results indicate that aberrant hypermethylation may be responsible for a significant proportion of epithelial ovarian tumours in which oestrogen receptor expression is lost.

Association of Functional Heme Oxygenase-1 Gene Promoter Polymorphism with Renal Transplantation Outcomes

Heme oxygenase-1 (HO-1) is a cytoprotective molecule and increased expression in experimental transplant models correlates with reduced graft injury. A functional dinucleotide repeat (GT)n polymorphism, within the HO-1 promoter, regulates gene expression; a short number of repeats (S-allele

Interleukin 18 promoter polymorphisms are not strongly associated with type 1 diabetes in a UK population

Interleukin 18 (IL18) is a proinflammatory cytokine whose levels are increased in the subclinical stage of insulin-dependent (type I) diabetes mellitus. Previous case-control studies have reported associations between IL18 -607C>A and -137G>C promoter polymorphisms and type I diabetes. We performed case-control and family-based association studies employing Pyrosequencing to assess if these IL18 polymorphisms are also associated with the development of type I diabetes in the Northern Ireland population. The chi2 analysis of genotype and allele frequencies for the IL18 polymorphisms in cases (n=433) vs controls (n=426) revealed no significant differences (P>0.05). Assessment of allele transmission distortion from informative parents to affected offspring also failed to confirm previously reported associations. Stratification of these analyses for age-at-onset and HLA-DR type did not reveal any significance associations. In conclusion, our data do not support the strong positive associations of IL18 promoter polymorphisms with type I diabetes reported in previous smaller studies.

Elafin prevents lipopolysaccharide-induced AP-1 and NF-kB activation via an effect on the ubiquitin-proteasome pathway

In this paper we follow on from our research into SLPI by assessing the immunomodulatory activity of elafin - an antiprotease related to SLPI and also present on the respiratory tract. We demonstrate for the first time that exogenously applied elafin inhibits lipopolysaccharide-induced activation of the NF-kappaB and AP-1 pathways in monocytes. I designed this project and supervised Marcus Butler during his MD thesis.

Ubiquitin-dependent proteolysis of trihydrophobin 1 (TH1) by the human papilloma virus E6-associated protein (E6-AP).

Human Papilloma virus E6-associated protein (E6-AP), which is known as an E3 ubiquitin ligase, mediates ubiquitination and subsequent degradation of a series of cellular proteins. In this paper, we identify here trihydrophobin 1 (TH1), an integral subunit of the human negative transcription elongation factor (NELF) complex, as a novel E6-AP interaction protein and a target of E6-AP-mediated degradation. Overexpression of E6-AP results in degradation of TH1 in a dose-dependent manner, whereas knock-down of endogenous E6-AP elevates the TH1 protein level. TH1 protein turnover is substantially faster, compared to controls, in cells that overexpressed E6-AP. Wild-type E6-AP promotes the ubiquitination of TH1, while a catalytically inactive point mutant of E6-AP abolishes its ubiquitination. Furthermore, in vitro ubiquitination assay also demonstrates that TH1 can be ubiquitinated by E6-AP. The degradation is blocked by treatment with proteasome inhibitor MG132. Herein, we provide strong evidence that TH1 is a specific substrate that is targeted for degradation through E6-AP-catalyzed polyubiquitination.

The radiation-inducible pE9 promoter driving inducible nitric oxide synthase radiosensitizes hypoxic tumour cells to radiation

Driving high-level transgene expression in a tumour-specific manner remains a key requirement in the development of cancer gene therapy. We have previously demonstrated the strong anticancer effects of generating abnormally high levels of intracellular NO• following the overexpression of the inducible nitric oxide synthase (iNOS) gene. Much of this work has focused on utilizing exogenously activated promoters, which have been primarily induced using X-ray radiation. Here we further examine the potential of the pE9 promoter, comprising a combination of nine CArG radio-responsive elements, to drive the iNOS transgene. Effects of X-ray irradiation on promoter activity were compared in vitro under normoxic conditions and various degrees of hypoxia. The pE9 promoter generated high-level transgene expression, comparable with that achieved using the constitutively driven cytomegalovirus promoter. Furthermore, the radio-resistance of radiation-induced fibrosarcoma-1 (RIF-1) mouse sarcoma cells exposed to 0.1 and 0.01% O2 was effectively eliminated following transfection with the pE9/iNOS construct. Significant inhibition of tumour growth was also observed in vivo following direct intratumoural injection of the pE9/iNOS construct compared to empty vector alone (P<0.001) or to a single radiation dose of 10?Gy (P<0.01). The combination of both therapies resulted in a significant 4.25 day growth delay compared to the gene therapy treatment alone (P<0.001). In summary, we have demonstrated the potential of the pE9/iNOS construct for reducing radio-resistance conferred by tumour cell hypoxia in vitro and in vivo, with greater tumour growth delay observed following the treatment with the gene therapy construct as compared with radiotherapy alone.

The tissue plasminogen activator gene promoter: a novel tool for radiogenic gene therapy of the prostate?

Radiation therapy is a treatment modality routinely used in cancer management so it is not unexpected that radiation-inducible promoters have emerged as an attractive tool for controlled gene therapy. The human tissue plasminogen activator gene promoter (t-PA) has been proposed as a candidate for radiogenic gene therapy, but has not been exploited to date. The purpose of this study was to evaluate the potential of this promoter to drive the expression of a reporter gene, the green fluorescent protein (GFP), in response to radiation exposure. METHODS: To investigate whether the promoter could be used for prostate cancer gene therapy, we initially transfected normal and malignant prostate cells. We then transfected HMEC-1 endothelial cells and ex vivo rat tail artery and monitored GFP levels using Western blotting following the delivery of single doses of ionizing radiation (2, 4, 6 Gy) to test whether the promoter could be used for vascular targeted gene therapy. RESULTS: The t-PA promoter induced GFP expression up to 6-fold in all cell types tested in response to radiation doses within the clinical range. CONCLUSIONS: These results suggest that the t-PA promoter may be incorporated into gene therapy strategies driving therapeutic transgenes in conjunction with radiation therapy.