Date of Award

2010

Document type

Thesis

Degree Name

PhD (Doctor of Philosophy)

First Supervisor

Professor Jochen Prehn

Second Supervisor

Dr Caoimhin Concannon

Keywords

Neoplasms, Cell Death, Heat-Shock Response

Abstract

Defects in apoptosis are one of the major causes of cancer. Several therapies have been developed to treat cancers such as chemotherapy, irradiation and hyperthermia. In the first part of this study an investigation of the underlying mechanism of lethal hyperthermia was undertaken in HeLa cervical cancer cells. A lethal heat shock activated the JNK pathway and increased p53, Puma, Bim, Mcl-I and Hsp70 expression. Caspase-3 was also activated but analysis of caspase- 2, caspase-8 and Bid cleavage showed little activation of these proteins making their requirement unclear. More importantly a robust Hsp70 induction was observed upon lethal hyperthermia which repressed amplified JNK phosphorylation and Bim expression. Interestingly inhibition of Hsp7O resulted in activation of Foxo3a, another protein involved in apoptosis. High levels of Hsp70 have been detected in several cancers and is involved in the resistance to anti-cancer treatments. Inhibition of the proteasome is another strategy used in cancer therapies but also results in Hsp70 up-regulation. We designed a reporter plasmid that mimics Hsp70 expression using the EGFP as a fluorescent probe. Studies investigating the stress dependent transcriptional expression of Hsp70 upon proteasome inhibition using epoxomicin were conducted in HCT 116 colon cancer cells at the single cell level using epifluorescent microscopy and High Content Screening. Increases in Hsp70 levels were evident in a stress dependent manner. Survival of cells upon proteasome inhibition was related to the amount of inducible Hsp70 accumulated which was dependent on two factors; the rate at which Hsp70 synthesis occurred and the length of activation of the Hsp70 promoter. Hsp70 basal expression and the onset time of its synthesis did not contribute to cell protection.

Creative Commons License

Creative Commons License
This work is licensed under a Creative Commons Attribution-Noncommercial-Share Alike 3.0 License.

File Size

9,700 KB

Comments

A thesis submitted to the Royal College of Surgeons in Ireland for the degree of Doctor of Philosophy from the National University of Ireland in 2010.

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