Small molecule inhibition of HDM2 leads to p53-mediated cell death in retinoblastoma cells

Purpose: To determine the efficacy of inducing p53-mediated cell death in retinoblastoma cells by Nutlin 3A, a small molecule HDM2 inhibitor. Methods: Retinoblastoma cell lines WER1- RB-1 and Y79 were treated with Nutlin 3A. Cell viability assays, Western blot analyses, confocal microscopy, and flow cytometry were performed to measure cell survival, p53 protein levels, activation of downstream targets, and apoptosis. To determine whether the effects of Nutlin 3A were p53- dependent, cell viability assays were performed on Y79 cells expressing short interfering RNA ( siRNA) against p53. Results: Nutlin 3A induced cell death in Y79 and WERI- RB- 1 in the 5- to 10- mu M dose range. Treated cells demonstrated increased protein levels of p53 and the p53 targets p21 and HDM2. Phosphorylation of p53- serine-15, a marker for activation of p53 via genotoxic mechanisms, was absent. Y79 cells expressing siRNA against p53 demonstrated resistance to Nutlin 3A. Conclusions: Nutlin 3A induced p53- mediated apoptosis in a dose-dependent, nongenotoxic fashion in 2 retinoblastoma cell lines. Clinical Relevance: Nutlin 3A is effective against retinoblastoma cells in a nongenotoxic manner. Because the mutagenic effects of radiation and chemotherapy may increase risks of secondary tumor formation, targeted p53 activation may be a safer alternative treatment for retinoblastoma in the future.