PO-490 Treatment of P53 wild-type cells with P53-inducer idasanutlin® (RG7388) generates stable drug-resistant clones

Abstract

IntroductionThe protein p53 is activated in response to DNA damage. The active protein induces cell cycle arrest and DNA repair program and thus protects the organism against carcinogenic events. Inactivation of p53 gives the cancer cell a profit of genetic instability, resulting in enhanced cancer cell microevolution. Apart from gaining direct mutations in p53-coding gene, overexpression of Mdm2 protein, which is a natural negative regulator of p53, is the most common method utilised by cancer to keep p53 inactive. Mdm2 binds to p53, blocks its transactivation domain and directs p53 to degradation. The re-activation of p53 protein by forced dissociation of Mdm2-p53 complexes using small molecule Mdm2 antagonists is currently believed to be a promising therapeutic strategy of the treatment of cancers expressing wild-type p53. Several representatives of this class of molecules, such as Idasanutlin (RG7388), are currently evaluated in clinical trials. However no data concerning the generation of secondary resistance is available for those modern, optimised compounds.Material and methodsHuman osteosarcoma U-2 OS cells were treated with Idasanutlin, followed by the analysis of viability (MTT assay), p53 activation (western blotting), the induction of apoptosis (annexin V staining) and cell cycle distribution (double staining with BrdU/FITC-anti-BrdU and propidium iodide). Homogenous populations of U-2 OS cells were established by expanding the monoclonal populations from single U-2 OS cells. For the generation of drug-resistant subpopulations the cells were treated with 5 µM Idasanutlin for 12–20 days. The resistant populations were visualised by in situ staining of the growing cells with BrdU/FITC-anti-BrdU and Hoechst 33 342.Results and discussionsThe treatment of U-2 OS cells with Idasanutlin leads to a strong induction of p53 expression level and the expression of p53-target gene encoding p21 protein. As a result Idasanutlin induces tremendous cell cycle arrest. However no apoptosis is induced and Idasanutlin -treated U-2 OS cells can be cultured for long time periods in the presence of the drug. This prolonged treatment of U-2 OS cells leads to de novo generation of cell populations that escape from the cytostatic effect evoked by Idasanutlin.ConclusionAlthough modern Mdm2 antagonists have much improved activities compared to their precursors (i.e. nutlin-3), they suffer from similar weaknesses, which are limited elimination of cancer cells and the generation of p53-mutated drug resistant subpopulations.