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Title: Global epigenetic profiling in malignant and normal urothelial cell lines
Author: Dudziec , Ewa
ISNI:       0000 0004 2742 994X
Awarding Body: University of Sheffield
Current Institution: University of Sheffield
Date of Award: 2011
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Epigenetic regulation of gene expression IS commonly altered in human cancer. Numerous alterations of DNA methylation and microRNA expression have been described in bladder cancer and these reflect the biology and phenotype of the disease. This common disease arises by distinct pathways with low and high grade differentiation. Epigenetic gene regulation is stabilized within a chromatin state by maintainers such as histone modification or DNA methylation. I hypothesized that epigenetic gene regulation reflects an interaction between histone and DNA modifications, and differences between normal and malignant urothelial cells represent carcinogenic events within bladder cancer. To test this hypothesis I profiled two histone modifications thought to be repressive (H3K9-3M and H3K27-3M) using ChIP-Seq, cytosine methylation using MeDIP and epG Isl and microarrays and mRNA expression in normal urothelial cells and cell lines that represent non invasive and invasive tumours. In genes with low expression I identified associated H3K27-3M and DNA methylation each in 20-30% of genes and both marks in 5% of genes. H3K9-3M was detected in 5-10% of genes but was not associated with overall expression. DNA methylation was more closely related to gene expression in malignant than normal cells. H3K27-3M was the epigenetic mark most specifically con-elated to gene silencing. I identified a panel of genes with cancer speci fie-epigenetic mediated aberrant expression including those with reported carcinogenic functions and members potentially mediating a positive epigenetic feedback loop. Pathway enrichment analysis revealed genes marked by H3K9-3M were involved with cell homeostasis, those marked by H3K27-3M mediated pro-carcinogenic processes and those marked with cytosine methylation were mixed in function. Hierarchical clustering revealed that gene panel stratified samples according to the presence and phenotype of bladder cancer. finally, the last part of my thesis concentrates on identifying epigenetic events associated with chemoresistance in bladder cancer. Currently, acquired resistance to drugs is the major obstacle in the successful treatment of advanced UCC. In addition, many tumours initially respond to chemotherapy, but subsequently develop secondary resistance. In order to identify epigenetic mechanisms associated with chemoresistance, I developed eisplatin resistant cell lines with 2 different approaches and investigated genome wide dysregulation of DNA methylation and microRNA expression in these. Resistant cells generated with different methodology and consequently with different stress applied, had very few common characteristics. They were found to have distinct profiles of microRNAs expression with more differentially expressed microRNAs (11=39) in the resistant cells derived from heterogeneous cell populations when compared to those from single cell clones (n=16). Only 8 microRNAs were shared between the resistant cells (including 5 with increased and 3 with decreased expression). Established resistant cells were also characterised by very distinct methylation profiles. The heterogeneous population of resistant cells were found to have the highest number of enriched and decreased in 5MC probes when compared to the cell lines generated from the single clones. Only 0.1 % of 5MC enriched and 0.02% 5MC decreased probes were shared between the established resistant cells. In summary, this Thesis illustrates that DNA methylation, histone modifications and microRNA expression are important regulatory mechanisms altered within UCc. These represent potential biomarkers and therapeutic targets that could be used to overcome treatment resistance owing to the reversible nature of their modifications.
Supervisor: Not available Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID:  DOI: Not available