The protein p53 protects the organism against carcinogenic events by the 
induction of cell cycle arrest and DNA repair program upon DNA damage. Virtually 
all cancers inactivate p53 either by mutations/deletions of the TP53 gene or by 
boosting negative regulation of p53 activity. The overexpression of MDM2 protein 
is one of the most common mechanisms utilized by p53wt cancers to keep p53 
inactive. Inhibition of MDM2 action by its antagonists has proved its anticancer 
potential in vitro and is now tested in clinical trials. However, the prolonged 
treatment of p53wt cells with MDM2 antagonists leads to the development of 
secondary resistance, as shown first for Nutlin-3a, and later for three other 
small molecules. In the present study, we show that secondary resistance occurs 
also after treatment of p53wt cells with idasanutlin (RG7388, RO5503781), which 
is the only MDM2 antagonist that has passed phase II and entered phase III 
clinical trials, so far. Idasanutlin strongly activates p53, as evidenced by the 
induction of p21 expression and potent cell cycle arrest in all the three cell 
lines tested, i.e., MCF-7, U-2 OS, and SJSA-1. Notably, apoptosis was induced 
only in SJSA-1 cells, while MCF-7 and U-2 OS cells were able to restore the 
proliferation upon the removal of idasanutlin. Moreover, idasanutlin-treated U-2 
OS cells could be cultured for long time periods in the presence of the drug. 
This prolonged treatment led to the generation of p53-mutated resistant cell 
populations. This resistance was generated de novo, as evidenced by the 
utilization of monoclonal U-2 OS subpopulations. Thus, although idasanutlin 
presents much improved activities compared to its precursor, it displays the 
similar weaknesses, which are limited elimination of cancer cells and the 
generation of p53-mutated drug-resistant subpopulations.