3353

Background: The tumour supressor gene TP53 is frequently mutated in human cancers. Previously we have shown mutations in the L2/L3 domains of the p53 protein to be associated with resistance to doxorubicin. During this work several novel mutations located within or in close proximity to the L2/L3 domains of the p53 protein were discovered. In this study we present a functional characterisation of these mutants. Some other mutants found in a similar prospective study focusing on mechanisms of resistance to epirubicin in metastatic breast cancer are also included. Materials and methods: cDNA carrying the various p53 mutants were cloned into the expression vectors pcDNA3.1V5 and pcDNA4/HisMax. The mutant proteins were expressed in a panel of p53 negative cell lines. Subcellular localisation of the p53 mutants was monitored by immunofluorescence. Analysis of the capability to oligomerise and efficiency of DNA binding were performed by immunoprecipitation and electromobility shift assay (EMSA), respectively. Results: Wildtype p53 and all the studied mutants were predominantly localised in the nucleus, but the percentage of nuclear localisation ranged from 60 to 90% between the mutants. IP assays revealed that all mutants remained capable of binding wildtype p53, and hence are probably capable of forming the tetrameric structure characteristic of active p53 in vivo. Discussion: None of the characterised p53 mutants showed any major loss of function in the performed in vitro assays. However there were differences between some of the mutants and wildtype p53, and we speculate that these differences may be significant for the capability of a tumour to resist chemotherapy in vivo.

[Proc Amer Assoc Cancer Res, Volume 47, 2006]