Phenotypic and proteomic analysis of 5-Fluorouracil treated normal and carcinoma cells

The anti-metabolite - 5-Fluorouracil (5-FU) - is the most widely used chemotherapeutic drug. It exerts its anti cancer effect through incorporation into DNA and RNA. Characterisation of this drugs mode of action is crucial in the development of future therapies. There have been many DNA microarray experiments performed in order to gain such information. However, only two proteomic experiments have been performed to date that look at the effect of 5-FU treatment. Here the proteomic alterations induced by IC8o 5-FU treatment of normal and cancer cells of epithelial origin of the lung and breast are investigated. These cell lines include a lung adenocarcinoma (A549), a non small cell lung carcinoma cell line (DLKP), normal bronchial epithelial cell line (NHBE), a breast adenocarcinoma (MCF-7) and human mammary epithelial cells (HMEC). Phenotypes were characterised and 5-FU was found to induce and reduce invasion in various cell lines. Adherence was altered in one of the three cancer cell lines to the extracellular matrix proteins collagen type IV and fibronectin. Differential regulated proteins were quantified using 2 dimensional difference gel electrophoresis (2D-DIGE) and differentially regulated proteins were identified using matrix-assisted laser desorption/ionization time of flight mass spectrometry (MALDI-ToF MS). Data shows the NHBE showed a dose dependent response to 5-FU treatment. Proteins that were found regulated are discussed in terms of microtubule dynamics and future drug combinations, translation control and stress, cytosketetal dynamics and invasion, and inhibition of apoptosis. 5-Fluorouracil is being increasing replaced in clinical trials with 3rd generation 5-FU drugs such as capecitabine. Capecitabine is converted in the liver to 5-fluoro-5’- deoxyuridine (55FdU) and this is converted to 5-FU by thymidine phosphorylase whose over expression is induced by radiotherapy. 5-FU is converted to 5-fluoro-2’- deoxyuridine in the cytoplasm. A comparison using 2D-DIGE between DLKP treated 5-FU and the fluoropyrimidines - 52FdU and 55FdU at ICgo cocentrations to determine similar proteomic alterations. Results demonstrated that the down regulation of Stathmin as a common fluoropyrimidine response. Furthermore this data may indicate a role for the use of vinca alkoloids or taxanes in combination with 5-FU. Resistance to 5-FU is a major clinical problem as mediated predominantly by over expression of Thymidylate Synthetase. A variant of DLKP was generated by pulse selection with 55FdU and showed a ~4 fold resistance to 5-FU. Proteomic analysis using 2D-DIGE identified several proteins involved in uracil metabolism and oxidative metabolism differentially regulated between DLKP and DLKP-55. DLKP is a heterogeneous cell line composed of at least three subpopulations. These subpopulations were isolated and were assigned the names DLKP-SQ, DLKP-I and DLKP-M. In this thesis data is presented demonstrating highly significant differences in motility and invasion between the clonal subpopulation. Analysis of proteomic alteration was carried out using 2D-DIGE on both the total cell lysate and the hydrophobic proteomes. Proteins were identified in the total cell lysates that suggest that DLKP-M is mesenchymal-like in nature as originally described and that the interconversion process observed between the clones is regulated at least by key proteins involved in protein metabolism. Analysis of the hydrophobic proteome found at least 300 proteins that correlated with motility/invasion and collagen synthesis. Identification of these proteins demonstrated increased association of microfilaments to the cellular membranes a process important in cellular motility. Furthermore 3 poorly described proteins were identified that correlated with motility/invasion and collagen synthesis. In addition data generated by these experiments indicates that fluoropyrimidine treatment of DLKP does not result in the selection of one of the subpopulations of DLKP and that DLKP-55 is not a subpopulation of DLKP. Data also shows the presence of protein in the heterogeneous population that are not present or are over expressed in the clonal populations indicating cell-cell communication.