Small molecule inhibition of HDM2 leads to p53-mediated cell death in retinoblastoma cells.
OBJECTIVE
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 WERI-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-microM 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.


CONCLUSIONS
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.
Elison
Jasmine R
JR
Department of Ophthalmology, Weill Medical College of Cornell University, New York Presbyterian Hospital, 520 E. 70th Street, New York, NY 10021, USA.
Cobrinik
David
D
Claros
Nidia
N
Abramson
David H
DH
Lee
Thomas C
TC
D017304:Annexin A5 / Q000378:metabolism; D017209:Apoptosis / Q000187:drug effects*; D015153:Blotting, Western; D002470:Cell Survival; D004305:Dose-Response Relationship, Drug; D005434:Flow Cytometry; D019084:Fluorescent Antibody Technique, Indirect; D006801:Humans; D007093:Imidazoles / Q000494:pharmacology*; D018613:Microscopy, Confocal; D010766:Phosphorylation; D010879:Piperazines / Q000494:pharmacology*; D051736:Proto-Oncogene Proteins c-mdm2 / Q000037:antagonists & inhibitors*; D034741:RNA, Small Interfering / Q000494:pharmacology; D019572:Retinal Neoplasms / Q000378:metabolism / Q000473:pathology*; D012175:Retinoblastoma / Q000378:metabolism / Q000473:pathology*; D014407:Tumor Cells, Cultured; D016159:Tumor Suppressor Protein p53 / Q000235:genetics / Q000378:metabolism*; D015854:Up-Regulation
D017304:Annexin A5; D007093:Imidazoles; D010879:Piperazines; D034741:RNA, Small Interfering; D016159:Tumor Suppressor Protein p53; C482205:nutlin 3; C497206:MDM2 protein, human; D051736:Proto-Oncogene Proteins c-mdm2