Human neuroblastoma cells with acquired resistance to the p53 activator RITA retain functional p53 and sensitivity to other p53 activating agents

Abstract

Adaptation of wild-type p53 expressing UKF-NB-3 cancer cells to the murine double minute 2 inhibitor nutlin-3 causes de novo p53 mutations at high frequency (13/20) and multi-drug resistance. Here, we show that the same cells respond very differently when adapted to RITA, a drug that, like nutlin-3, also disrupts the p53/Mdm2 interaction. All of the 11 UKF-NB-3 sub-lines adapted to RITA that we established retained functional wild-type p53 although RITA induced a substantial p53 response. Moreover, all RITA-adapted cell lines remained sensitive to nutlin-3, whereas only five out of 10 nutlin-3-adapted cell lines retained their sensitivity to RITA. In addition, repeated adaptation of the RITA-adapted sub-line UKF-NB-3(r)RITA(10 μM) to nutlin-3 resulted in p53 mutations. The RITA-adapted UKF-NB-3 sub-lines displayed no or less pronounced resistance to vincristine, cisplatin, and irradiation than nutlin-3-adapted UKF-NB-3 sub-lines. Furthermore, adaptation to RITA was associated with fewer changes at the expression level of antiapoptotic factors than observed with adaptation to nutlin-3. Transcriptomic analyses indicated the RITA-adapted sub-lines to be more similar at the gene expression level to the parental UKF-NB-3 cells than nutlin-3-adapted UKF-NB-3 sub-lines, which correlates with the observed chemotherapy and irradiation sensitivity phenotypes. In conclusion, RITA-adapted cells retain functional p53, remain sensitive to nutlin-3, and display a less pronounced resistance phenotype than nutlin-3-adapted cells.

Figure 1

p53 activation in A76T p53-mutated UKF-NB-3^rRITA^10 μMIV cells. Cells were treated with RITA (10 μM) or nutlin-3 (10 μM) for 24 h and investigated by western blot for the expression of p53 target genes

Figure 2

Influence of RITA and nutlin-3 on p53 activation and apoptosis in UKF-NB-3, UKF-NB-3^rNutlin^10μM, and UKF-NB-3^rRITA^10μM cells. (a–b) Cells were treated with RITA (10 μM) or Nutlin-3 (10 μM) for 24 h and investigated by western blot for the expression of p53 target genes (a) or for the formation of the cleaved form of PARP (cPARP) (b). β-actin served as loading control. (c) Cells were treated with RITA (10 μM) or Nutlin-3 (10 μM) for 24 h. Caspase 3 activation was determined by Caspase-Glo 3/7 Assay. *P<0.05

Figure 3

Expression of apoptosis-associated proteins in UKF-NB-3, UKF-NB-3^rNutlin^10 μM, and UKF-NB-3^rRITA^10 μM cells

Figure 4

Hierarchical cluster analysis based on transcriptomics data of UKF-NB-3 and UKF-NB-3 sub-lines adapted to RITA- or nutlin-3. Each cell line was analysed in triplicate