Activation of p53 occurs by stabilization and posttranslational modifications in response to stress signals. IEC-6 cells expressed low basal levels of p53, which increased in response to CPT, leading to the increased expression of Bax and apoptosis (
Fig. 2A). Conversely, confluent serum-starved ARPE-19 cells had high basal expression of p53 and p21Cip1 proteins (
Fig. 2A). CPT robustly increased p53 protein levels and phosphorylation in both cell types (
Fig. 2A). However, it failed to induce p53-dependent expression of PUMA, Noxa, and Bax (
Figs. 8B,
9B), which caused mitochondrial depolarization. Therefore, no effect was observed on caspase-9 and -3 activation in ARPE-19 cells. Different results were reported by Nair et al.,
13 who found that 0.1 μM CPT for 48 hours resulted in caspase-3 activation and cell death in ARPE-19 cells. The difference in the response of cells to CPT between our findings and those of Nair et al.
13 appeared to be due to differences in cell cycle status. Serum-starved, quiescent, confluent ARPE-19 cells treated with 20 μM CPT for varying times failed to undergo apoptosis. However, cells treated with a low concentration of CPT (5 μM) at the time of plating (during proliferation) significantly increased mean doubling time (
Fig. 3A). In addition, low doses of CPT significantly induced p53 and its downstream target, p21Cip1, indicating the predominant effects were on cell cycle regulators rather than on apoptotic inducers (
Fig. 3B). Others have found that 100 μM CPT treatment for 4 hours failed to induce significant apoptosis, and prolonged exposure for 72 hours was required to induce significant cytotoxicity.
51 The confluent, serum-starved, quiescent ARPE-19 monolayers used in our model are more representative of those found in an intact retina. Under normal physiologic conditions, primary RPE cells are constantly exposed to oxidant-mediated injuries, causing a mild, stresslike increase of p53-dependent signaling that may prime these cells to be protected from the toxicity of oxidative stress-induced cell death. Preconditioning ARPE-19 cells with sublethal doses of hydrogen peroxide increased the cellular resistance to subsequent toxic exposures.
52 Thus, it is reasonable to posit that increased expression of p53 and p21Cip1 in ARPE-19 cells is crucial to modulate proliferation and to safeguard these cells from external stress. Both cell cycle arrest and senescence require complete engagement of p53 and Rb-mediated signaling pathways.
53 p53-Mediated induction of p21Cip1 inhibits the cyclin-dependent kinase activity required for the phosphorylation of Rb protein, which releases bound E2F and leads to the resumption of cell cycle progression.
40,41 Our results demonstrate that a low dose of Nutlin-3 (5 μM) for 24 hours upregulates p53 and causes a block in cell proliferation, primarily because of the accumulation of the Cdk inhibitor p21Cip1, which decreases levels of phosphorylated Rb (
Fig. 3B). Nutlin-3 affects senescence in mouse fibroblasts
54 and proliferation and differentiation in preosteoclasts
55 by a similar p53-dependent mechanism.