Inhibition of Mdm2 sensitizes human retinal pigment epithelial cells to apoptosis

Sujoy Bhattacharya, Ramesh M. Ray, Edward Chaum, Dianna A. Johnson, Leonard R. Johnson

Research output: Contribution to journalArticle

10 Citations (Scopus)

Abstract

Purpose. Because recent studies indicate that blocking the interaction between p 53 and Mdm2 results in the nongenotoxic activation of p 53, the authors sought to investigate whether the inhibition of p 53-Mdm2 binding activates p 53 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 p 53 from Mdm2 and, thus, to increase p 53 activity. Knockdown of p 53 expression was accomplished by using p 53 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 p 53 in response to Nutlin-3 also increased levels of Noxa, p 53-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 p 53 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 p 53 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 p 53 binding to Mdm2 frees a pool of p 53 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.

Original languageEnglish (US)
Pages (from-to)3368-3380
Number of pages13
JournalInvestigative Ophthalmology and Visual Science
Volume52
Issue number6
DOIs
StatePublished - May 1 2011

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Retinal Pigments
Epithelial Cells
Apoptosis
Camptothecin
DNA Fragmentation
Cycloheximide
Caspase 3
Noxae
Caspase 9
DNA
Caspases
Cell Cycle Checkpoints
Small Interfering RNA
Up-Regulation
nutlin 3

All Science Journal Classification (ASJC) codes

  • Ophthalmology
  • Sensory Systems
  • Cellular and Molecular Neuroscience

Cite this

Inhibition of Mdm2 sensitizes human retinal pigment epithelial cells to apoptosis. / Bhattacharya, Sujoy; Ray, Ramesh M.; Chaum, Edward; Johnson, Dianna A.; Johnson, Leonard R.

In: Investigative Ophthalmology and Visual Science, Vol. 52, No. 6, 01.05.2011, p. 3368-3380.

Research output: Contribution to journalArticle

Bhattacharya, Sujoy ; Ray, Ramesh M. ; Chaum, Edward ; Johnson, Dianna A. ; Johnson, Leonard R. / Inhibition of Mdm2 sensitizes human retinal pigment epithelial cells to apoptosis. In: Investigative Ophthalmology and Visual Science. 2011 ; Vol. 52, No. 6. pp. 3368-3380.
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abstract = "Purpose. Because recent studies indicate that blocking the interaction between p 53 and Mdm2 results in the nongenotoxic activation of p 53, the authors sought to investigate whether the inhibition of p 53-Mdm2 binding activates p 53 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 p 53 from Mdm2 and, thus, to increase p 53 activity. Knockdown of p 53 expression was accomplished by using p 53 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 p 53 in response to Nutlin-3 also increased levels of Noxa, p 53-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 p 53 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 p 53 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 p 53 binding to Mdm2 frees a pool of p 53 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.",
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AB - Purpose. Because recent studies indicate that blocking the interaction between p 53 and Mdm2 results in the nongenotoxic activation of p 53, the authors sought to investigate whether the inhibition of p 53-Mdm2 binding activates p 53 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 p 53 from Mdm2 and, thus, to increase p 53 activity. Knockdown of p 53 expression was accomplished by using p 53 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 p 53 in response to Nutlin-3 also increased levels of Noxa, p 53-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 p 53 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 p 53 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 p 53 binding to Mdm2 frees a pool of p 53 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.

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