ROLES OF INOSITOL DIPHOSPHATES IN DNA REPAIR AND EFFECTS OF ASPIRIN ANALOGUES ON OESOPHAGEAL CANCER
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AuthorsKilari, Rajagopal Sharada.
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AbstractInositol phosphates (IPs) are important signalling molecules with various biological roles in a cell. One such role it is often associated with is DNA repair. The DNA repair process following DNA insult is considered crucial for the genomic integrity and stability. Failure to perform this task will result in mutations and possibly disease. Thus, it is important that we expand our knowledge on how these repair processes occur and identify the key factors involved in its regulation. The aim of this project was to investigate whether DNA repair was mediated by inositol diphosphates (IDPs). Using a family of yeast knockout mutants with modulated levels of IPs, it was found that IDPs are crucial in repair of DNA following insult with bleomycin and 5-fluorouracil. The observed sensitivity of the mutants was thought to be due to lack of functional repair protein, UDG-like or APE-like, in the absence of essential cofactor such as IDPs. Experiments conducted revealed that the hypersensitive kcs1Δ contain both the repair proteins required to process the DNA lesions. However, extreme extraction methods were required to access these proteins, suggesting that the proteins are mislocalised and unavailable to access the damage site and perform DNA repair. GFP-tagging the proteins Ung1, Apn1 and Rad52 in kcs1Δ proved to be of little use as it failed to show exact localisation, movement and functionality status of these proteins following bleomycin insult. The enzymes accountable for the dephosphorylation of the IDPs in vivo are the diphosphoinositol polyphosphate phosphohydrolases (DIPPs). Little is known regarding the Michaelis-Menten kinetics parameters for Ddp1p/DIPPs. In this study, using improved methods for the enzymatic synthesis and electrophoretic purification of 1-InsP7, 5-InsP7 and InsP8, the DIPP family has been kinetically characterised. Each DIPP was found to ii display similar Km values for every substrate tested (range: 35-148 nM). The rank order of Kcat values (1-InsP7 > 5-InsP7 = InsP8) was identical for each enzyme, although DIPP-1 activity was observed to be 10- to 60-fold more than DIPP-2α/β and DIPP-3α/β, irrespective of the substrate. This study reveals that Ddp1, the yeast DIPP, is capable of hydrolysing not only 5-InsP7 but also 1-InsP7 and InsP8 to a single product, InsP6. The HPLC data found InsP7 accumulation to be relatively little during InsP8 breakdown by DIPPs. Such low build-up was found to be due to rapid conversion of InsP7 to InsP6. Through this study it is also clear that InsP8 prefers to dephosphorylate through 1-InsP7. In contrary, metabolically and functionally significant steady-state route of InsP8 synthesis was observed to be via 5-InsP7. Oesophageal cancer is considered as one of the deadliest cancers worldwide because of its aggressive nature and low survival rate. Epidemiologic studies have shown that low-dose daily intake of aspirin can decrease the incidence of oesophageal cancer. The data presented in this study show the effects of a number of in-house synthesized novel aspirin analogues on oesophageal cancer cell lines, squamous cell carcinoma (SSC) and adenocarcinoma (ADC). The aspirin analogues, fumaryldiaspirin (PN517) and benzoylsalicylates (PN524, PN528 and PN529), were observed to be more potent against the oesophageal cell lines than aspirin itself. Both, quantitative and qualitative apoptosis experiments conducted revealed that these compounds largely induced apoptosis, although some necrosis was evident with PN528 and PN529. Failure to recover following the treatment with these analogues emphasized that these drugs are largely cytotoxic in nature. The SSC cells (oe21) displayed increased sensitivity to the aspirin analogues compared to the ADC cell lines (flo-1 and oe33). The anticancer properties of these novel aspirin compounds appear to not involve the COX-enzymes at the tested concentrations. These initial findings support further studies into the potential of these aspirin analogues as chemotherapeutic agents against oesophageal cancer.
DescriptionA thesis submitted in partial fulfilment of the requirements of the University of Wolverhampton for the degree of Doctor of Philosophy.