An Investigation of the Role of Thymidine Phosphorylase in the Activation of 5-Fluorouracil in Colon Tumour Cell Lines

Medicine, Cellular biology, Oncology

Milne, Lesley Helen

University:University of Glasgow

关键词: Medicine, Cellular biology, Oncology;   

Others  :  http://theses.gla.ac.uk/74518/1/10992137.pdf

来源: University of Glasgow

【 摘 要 】

Thymidine phosphorylase (TP) is identical to platelet-derived endothelial cell growth factor (PD-ECGF) and was originally isolated from platelets as the sole moiety with endothelial cell mitogenic activity. TP activity is increased in a number of tumour types compared with adjacent normal tissues including colon, stomach and breast cancer. TP has a role in tumour angiogenesis and increased activity in tumours confers a more aggressive tumour phenotype. TP also has a role in the activation of the fluoropyrimidine 5-fluorouracil (5-FU) to 5-fluorodeoxyuridine (5FdUrd). Intratumoural expression of TP is heterogeneous and may explain why only a proportion of patients respond to therapy with 5-FU. Modulation of 5-FU with interferon-alpha (IFN-alpha) increases the cytotoxicity of 5-FU in colon cancer cell lines. This is thought to occur through an increase in the activity of TP leading to greater formation of 5'-fluorodeoxyuridine monophosphate (FdUMP) and increased inhibition of thymidylate synthase (TS). HT-29 colon adenocarcinoma cell lines were transfected with TP cDNA and a novel TP activity assay demonstrated a 5-fold increase in TP activity in HT-29(TP) cells compared with the parental cells and the vector alone transfected controls, HT-29(V). 5-FU cytotoxicity was increased 1.6-fold in HT-29(TP) cells compared with HT-29(V) cells (p<0.001, Students' t-test). There was no significant difference in cytotoxicity between HT-29(TP) and HT-29 parental cells (p>0.1, Students' t-test). Transfection alone without TP cDNA increased the resistance of HT-29 cells to 5-FU. The apparent increase in 5-FU cytotoxicity in HT-29(TP) cells was not enhanced by Leucovorin (LCV) or Deoxyinosine and cytotoxicity was not significantly reversed by exogenous thymidine, although a small increase in 5-FU IC50 was observed in the controls. Inhibition of TS by FdUMP was observed in these cells; however it does not appear to be the critical mechanism of action of 5-FU. The data from this thesis suggest that an increase in the activity of TP alone (5-fold) is not sufficient to significantly increase 5-FU cytotoxicity. Confirmation was obtained when TP activity and 5-FU cytotoxicity were measured in a number of cell lines from several tumour types. TP activity alone did not determine the sensitivity of the cell lines to 5-FU. Despite disappointing results with respect to 5-FU, a greater and more significant effect may be observed with 5-FU pro-drugs such as 5-dFUrd and Tegafur, which require activation by TP. The application of TP as a candidate gene for gene-directed/fluoropyrimidine pro-drug therapy is therefore promising as a means of improving the current lack of effective treatment for colon cancer and requires further investigation.