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Title: A proteomic approach to identifying novel determinants of tumour response to chemotherapy in colorectal cancer
Author: Wilson, P. M. D.
ISNI:       0000 0001 3570 4567
Awarding Body: Queens -Belfast
Current Institution: Queen's University Belfast
Date of Award: 2008
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Colorectal cancer (CRC)is the leading cause of cancer-related deaths in the western world. In the metastatic setting, less than half of patients who undergo chemotherapy will receive any , ...' benefit from treatment. A major factor limiting the 'effectiveness of chemotherapy is .acquired or inherent drug-resistance. We have developed a panel of 5-fluorouracil- (5-FU-), oxaliplatinand CPT-ll-resistant HCT1l6 CRC cell lines and using 2D electrophoresis in combination with MALDI-TOF mass spectrometry, analysed differences in basal protein expression in parental and drug-resistant cells. Furthermore, we have validated these data using real-time RT-PCR and Western blotting techniques. We have demonstrated modulation of a number of cytosolic and nuclear matrix proteins in cells resistant to oxaliplatin, 5-FU and CPT-ll. We identified the calcium-binding protein calretinin as being significantly over-expressed at both the protein and mRNA level in the p53 wild-type HCTl16 oxaliplatin- and 5-FU-resistant cells compared to parental ,cells. Furthermore, treatment with 5-FU, oxaliplatin and SN38 ~esulted in potent induction of calretinin mRNA expression in parental cells with no concurrent increase in protein. Ttreatment in oxalipla~in and SN38-resistant cells demonstrated a marked reduction in levels of induction, suggesting that elevated calretinin mRNA expression is associated with 'drug-induced cytotoxicity. Augmented calretinin induction following drug treatment in HCTl16 p53 null cells indicated that p53 plays a pivotal role in regulating calretinin mRNA induction in response to 5-FU, oxaliplatin and SN38. The mechanism by which p53 regulates calretinin mRNA expression remains to be elucidated. Further studies demonstrated that stable over-expression of calretinin sensitised p53 wild-type HCT1l6 parental cells to sub-optimal concentrations of 5-FU, oxaliplatin and SN38 as determined by MTT, cell cycle and PARP cleavage analysis. In addition, down-regulation of calretininusing siRNA oligonucleotides conferred a protective effect following treatment with 5-FU and oxaliplatin. Collectively, these results suggest that calretinin plays a pivotal role in modulating cytoxic drug-induced apoptosis in-vitro.
Supervisor: Not available Sponsor: Not available
Qualification Name: Queens -Belfast, 2008 Qualification Level: Doctoral
EThOS ID:  DOI: Not available