title: Inhibition of Mdm2 sensitizes human retinal pigment epithelial cells to apoptosis.
abstract: OBJECTIVE
  Because recent studies indicate that blocking the interaction between p53 and Mdm2 results in the nongenotoxic activation of p53, the authors sought to investigate whether the inhibition of p53-Mdm2 binding activates p53 and sensitizes human retinal epithelial cells to apoptosis.
  
  
  METHODS
  Apoptosis was evaluated by the activation of caspases and DNA fragmentation assays. The Mdm2 antagonist Nutlin-3 was used to dissociate p53 from Mdm2 and, thus, to increase p53 activity. Knockdown of p53 expression was accomplished by using p53 siRNA.
  
  
  RESULTS
  ARPE-19 and primary RPE cells expressed high levels of the antiapoptotic proteins Bcl-2 and Bcl-xL. Exposure of these cells to camptothecin (CPT) or TNF-α/ cycloheximide (CHX) failed to induce apoptosis. In contrast, treatment with the Mdm2 antagonist Nutlin-3 in the absence of CPT or TNF-α/CHX increased apoptosis. Activation of p53 in response to Nutlin-3 also increased levels of Noxa, p53-upregulated modulator of apoptosis (PUMA), and Siva-1, decreased expression of Bcl-2 and Bcl-xL, and simultaneously increased caspases-9 and -3 activities and DNA fragmentation. Knockdown of p53 decreased the basal expression of p21Cip1 and Bcl-2, inhibited the Nutlin-3-induced upregulation of Siva-1 and PUMA expression, and consequently inhibited caspase-3 activation.
  
  
  CONCLUSIONS
  These results indicate that the normally available pool of intracellular p53 is predominantly engaged in the regulation of cell cycle checkpoints by p21Cip1 and does not trigger apoptosis in response to DNA-damaging agents. However, the blockage of p53 binding to Mdm2 frees a pool of p53 that is sufficient, even in the absence of DNA-damaging agents, to increase the expression of proapoptotic targets and to override the resistance of RPE cells to apoptosis.
authors.affiliation: Department of Physiology, University of Tennessee Health Science Center, Memphis, TN 38163, USA. sbhatta3@uthsc.edu
authors.forename: Sujoy
authors.initials: S
authors.lastname: Bhattacharya
authors.forename: Ramesh M
authors.initials: RM
authors.lastname: Ray
authors.forename: Edward
authors.initials: E
authors.lastname: Chaum
authors.forename: Dianna A
authors.initials: DA
authors.lastname: Johnson
authors.forename: Leonard R
authors.initials: LR
authors.lastname: Johnson
mesh_terms: D000328:Adult; D017209:Apoptosis / Q000187:drug effects*; D015153:Blotting, Western; D053148:Caspase 3 / Q000378:metabolism; D053453:Caspase 9 / Q000378:metabolism; D002460:Cell Line; D049109:Cell Proliferation / Q000187:drug effects; D053938:DNA Fragmentation; D006801:Humans; D007093:Imidazoles / Q000494:pharmacology*; D010879:Piperazines / Q000494:pharmacology*; D011485:Protein Binding / Q000187:drug effects; D019253:Proto-Oncogene Proteins c-bcl-2 / Q000037:antagonists & inhibitors; D051736:Proto-Oncogene Proteins c-mdm2 / Q000037:antagonists & inhibitors*; D034741:RNA, Small Interfering / Q000494:pharmacology; D055213:Retinal Pigment Epithelium / Q000378:metabolism / Q000473:pathology*; D014162:Transfection; D016159:Tumor Suppressor Protein p53 / Q000378:metabolism*; D051020:bcl-X Protein / Q000037:antagonists & inhibitors
chemical_list: C494811:BCL2L1 protein, human; D007093:Imidazoles; D010879:Piperazines; D019253:Proto-Oncogene Proteins c-bcl-2; D034741:RNA, Small Interfering; D016159:Tumor Suppressor Protein p53; D051020:bcl-X Protein; C482205:nutlin 3; C497206:MDM2 protein, human; D051736:Proto-Oncogene Proteins c-mdm2; D053148:Caspase 3; D053453:Caspase 9