Drug resistance, and the role of p53, in lung cancer cell lines

This thesis sets out to increase our knowledge of mechanisms by which lung cancer cells develop resistance to chemotherapeutic agents. The involvement of the tumour suppressor p53 in the development of drug resistance in lung cancer cell lines was investigated. p53 is a tumour suppressor gene, which is mutated in more than half of all tumours. Most chemotherapeutic drugs cause DNA damage that is sensed by p53, which either arrests the cell cycle to allow DNA repair or induces apoptosis. Wildtype p53 was transfected into several cell lines; A549, which expresses wild-type p53, DLKP-SQ, which expresses mutant p53 and HI299 which is p53 null. Clones were isolated from these cell lines and tested for changes in sensitivity to the chemotherapeutic agents adriamycin, taxol and carboplatin. A549 transfected clones showed an increase in p53 protein after transfection, however, these cells did not display substantial changes in sensitivity to the chemotherapeutic agents tested. The cell line H1299, which is p53 null, expressed p53 protein after stable transfection, and was chosen for further analysis. This p53-expressing cell line displayed only small changes in sensitivity to chemotherapeutic agents compared to control-transfected cells. No correlation was observed between the p53 status of the cell lines and the ease of developing resistant variants. To further investigate the molecular basis of drug resistance in lung cancer, a panel of four cell lines were chosen for pulse-selection with chemotherapeutic agents. The cell lines include two adenocarcinoma (AC) and two large cell carcinoma (LCC) cell lines. They were pulse-selected with taxol and carboplatin, both of which are clinically relevant drugs for treatment of this type of cancer. The eight novel cell lines obtained were tested for sensitivity to a cross-section of chemotherapeutic agents and for expression of the multidrug resistance (MDR) efflux pump proteins, Pgp and MRP-1. The taxol-selected variants were chosen for further analysis because the resistance profile was stable and the concentration of drug used for selection was at a clinically achievable level. Microarray analysis was used to identify genes associated with the development of taxol resistance. Ten differentially expressed genes with a possible role in taxol resistance were chosen from this analysis. These genes were further investigated by siRNA transfection, in order to determine the functional relevance of these genes in drug resistance. The taxol-selected variants analysed by microarray analysis provide a unique opportunity to study less well characterised mechanisms of taxol resistance, since these variants do not display classical MDR