p53 can be degraded in the nucleus. (A) p53, HDM2, and proteasome subcellular localization in normal human fibroblasts (WS1). Cells were left untreated (unt), or treated with 20 nM LMB for 6 h, 640 ng/ml NCS for 4 h, or PIs for 4 h, and stained with antibodies to p53, HDM2, and the proteasome. (B) p53 and HDM2 nuclear:cytoplasmic (N/C) ratios for immunofluorescently stained WS1. See Materials and methods for determination of p53 and HDM2 N/C ratios. p53 ratios are depicted by gray bars and HDM2 ratios by white bars. Values represent the mean (±s.d.) of >30 cells. (C) Immunoblots of p53, HDM2, p53 phosphorylated S15 (PS15), and p53 acetylated lysine 382 (AcK382). WS1 cells were treated as in (A), then immunoblotted with antibodies to p53 (DO-1), HDM2 (IF2), and actin. The membrane was stripped and re-probed with an antibody to p53 PS15. Separate samples were lysed in acetylation lysis buffer, then stained with an antibody to AcK382. (D) p53 target gene induction. cDNA was harvested from WS1 that were untreated; LMB, NCS, or PI-treated (see (A)); or UV irradiated. HDM2 (white bars) or p21 (gray bars) gene induction was measured by real-time quantitative PCR. Induction is expressed as fold induction relative to untreated. Error bars indicate the 95% CI of ≥3 experiments. (E) Degradation of cytoplasmic p53 by HDM2. 2KO or SaOS-2 cells (data not shown) were transfected with plasmids containing luciferase, GFP, 150 ng p53 (wt, p53RN, NES, or NRN), and 1.2 μg HDM2 (wt) or empty vector (pcDNA3, (−)). Equal luciferase units of transfected cell lysates were immunoblotted with antibodies to p53 or GFP. (F) Densitometric analysis of p53 Western blots. Percent p53 remaining was calculated as the ratio of p53 co-transfected with wt HDM2 to p53, with empty vector normalized to GFP. Bars represent the mean band intensity±1 s.d. for >3 experiments.

Multiple stresses decrease HDM2 half-life in normal human fibroblasts. (A) HDM2 half-life in NCS-treated WS1 cells. WS1 were left untreated or treated with 640 ng/ml NCS for 4 h. Protein synthesis was inhibited by concurrent treatment with CHX for the indicated times. Steady-state levels of HDM2 are higher in stressed cells due to increased p53-dependent gene activation; therefore, for comparison of the half-lives of HDM2 with and without DNA damage, short and long film exposures (short exp. and long exp.) are shown. This figure is representative of >3 experiments. (B) Quantitation of NCS-induced HDM2 destabilization in (A). HDM2 band intensity was normalized to actin, then expressed relative to the t=0 controls. Each decrease of one unit of log₂ (band intensity) is equivalent to one half-life. (C) HDM2 half-life in UV-irradiated WS1 cells. WS1 were left untreated or exposed to 26 J/m² UVC irradiation 16 h before incubation with CHX for the indicated times. PIs were added to UV-irradiated cells 12 h after UV exposure, then cells were harvested 4 h later. (D) Quantitation of UV-induced HDM2 destabilization in (C). The blot was scanned and analyzed as in (B). (E) HDM2 half-life in BCNU-treated WS1 cells. WS1 were left untreated (data not shown) or incubated with 100 μM BCNU 8 h prior to incubation with CHX for the indicated times.

The HDM2 RING domain is required for DNA-damage-induced destabilization. U2OS cells were transfected with 2 μg of HDM2 RING domain mutant C464A and 125 ng GFP. Transfected cells were treated with 640 ng/ml NCS, then CHX for the indicated times. Untransfected cells were left untreated or incubated in NCS.

Proteasome inhibition prevents p53 activation by DNA damage. (A) HDM2 and p21 gene induction in PI-treated cells. WS1 cells were left untreated (bar 1), or treated with 50 ng/ml NCS for 1, 2, or 4 h (bars 2–4), PI alone for 4 h (bar 5), PI followed by concurrent treatment with NCS for 1, 2, or 4 h (bars 6–8), wortmannin (wort) alone for 30 min (bar 9), or wortmannin followed by concurrent treatment with NCS for 2 h (bar 10). RNA was harvested and subjected to real-time quantitative PCR as in Figure 1. Bars indicate the 95% CI of the mean induction of HDM2 (white) or p21 (gray). (B) p53 S15 phosphorylation status in cells treated with PI and NCS. WS1 cells were treated as in (A), then lysates were run on a 4–12% gradient gel and immunoblotted with antibodies to HDM2, phosphorylated p53 S15 (PS15), and actin, then stripped and re-probed with an antibody to unmodified p53 (DO-1). (C) HDM2/p53 association in PI and NCS-treated cells. One mg of WS1 or SaOS-2 (p53 (−)) whole-cell extract was immunoprecipitated with an HDM2 antibody (IF2, or SMP14 or 4B2, data not shown), then immunoblotted with antibodies to HDM2 and p53 (Ab-7). The blot was stripped and re-probed with p53 S15 antibody. (D) HDM2 and p21 gene induction in Nutlin-treated cells. WS1 cells were incubated in 10 μM Nutlin-3a (Nut3a) or 3b for 4 h, then PI for 4 h, then 50 ng/ml NCS for 3 h. Gene induction was analyzed as in (A). (E) HDM2/p53 association in Nutlin-treated cells. WS1 cells were treated as in (D), then 700 μg of whole-cell extract was immunoprecipitated with antibodies to HDM2 (IF2) or nonspecific IgG and immunoblotted with antibodies to HDM2 and p53 as in (C).

DNA damage destabilizes HDM2. (A) p53 and HDM2 immunofluorescence staining in transfected U2OS. Cells were co-transfected with 200 ng p53 and 1.6 μg HDM2, and left untreated (unt) or treated with PI or 640 ng/ml NCS for 4 h. Yellow cells in the ‘merge' panels indicate cells expressing both p53 and HDM2. Bar=20 μm. (B) Quantitation of p53 and HDM2 nuclear co-incidence. Cells with nuclear p53 staining were counted for the presence (HDM2+, yellow bars) or absence (HDM2−, red bars) of HDM2 co-expression, and displayed as a percentage of the total number of p53-positive cells. Greater than 347 cells were counted for each condition. (C) Half-life of HDM2 in transfected cells. U2OS cells were transfected with 2 μg HDM2 and 125 ng GFP. Transfected cells were treated with 640 ng/ml NCS for 4 h, then concurrently with carrier (t=0) or cycloheximide (CHX) for the indicated times. This figure is representative of >3 experiments. (D) Quantitation of HDM2 destabilization in (C). HDM2 band intensity was normalized to GFP, then normalized to the t=0 controls. Each decrease of one unit of log₂ (band intensity) is equivalent to one half-life.

HDM2 destabilization is regulated by PI 3-kinase related kinases. (A) Half-life of HDM2 ATM phosphorylation site mutants. U2OS cells were transfected with 2 μg of HDM2 (S395A, S395D, or wt) or pcDNA3 and 125 ng GFP. Transfected cells were treated with 640 ng/ml NCS or PI for 4 h, then CHX for the indicated times. Different exposures of each transfection are shown to facilitate comparison of HDM2 stability. (B) Quantitation of HDM2 destabilization in (A) and Figure 2C. HDM2 band intensity was normalized to GFP, then normalized to the t=0 controls. (C) Half-life of HDM2 in wortmannin-treated cells. Normal human fibroblasts (WS1) were left untreated or were treated with 50 μM wortmannin for 30 min, 50 ng/ml NCS for 2 h, or wortmannin, then NCS. Carrier (t=0) or CHX was added for the indicated times. This figure is representative of three experiments. (D) Quantitation of HDM2 destabilization in (C). HDM2 band intensity was normalized to actin, then normalized to the t=0 controls. Each decrease of one unit of log₂ (band intensity) is equivalent to one half-life.

HDM2 destabilization correlates temporally with p53 transcriptional activity. (A) Timecourse of ATM and p53 phosphorylation. WS1 cells were treated with 50 ng/ml NCS for the indicated times. Lysates was separated on a 3–8% Tris–acetate gel and Western blots were probed with antibodies to p53-phosphorylated S15 (PS15), HDM2, phosphorylated ATM (P-ATM), and actin. A second 4–12% Tris–glycine gel was immunoblotted and probed with antibodies to p21 and actin (data not shown). (B) Timecourse of p53 target gene activation in NCS-treated WS1 cells. Cells were treated with 50 ng/ml NCS for the indicated times, and real-time quantitative PCR was performed with primers to HDM2 (white) and p21 (gray). Bars represent one s.d. of mean gene induction normalized to untreated. (C) Half-life of phosphorylated S15 p53 at early and late treatments of NCS. WS1 cells were treated with 50 ng/ml NCS and CHX for the indicated times, then Western blotted with antibodies to phosphorylated p53 S15 and actin (data not shown). To compare the steady-state p53 levels, this image was compiled from one film exposure. (D) Timecourse of p53 and HDM2 stability. WS1 cells were treated with 50 ng/ml NCS and CHX for the indicated times, then lysates were immunoblotted with antibodies to unmodified p53 (DO-1), HDM2, and actin (data not shown). This figure is representative of three experiments. As p53 and HDM2 steady-state levels varied at different times of NCS treatment, images from films exposed for different time periods are shown to better compare p53 and HDM2 half-lives (see Figures 4 and 5). (E) Quantitation of p53 half-life in (D). p53 band intensity was normalized to actin (data not shown), then normalized to the t=0 controls. Each decrease of one unit of log₂ (band intensity) is equivalent to one half-life. (F) Quantitation of HDM2 half-life in (D). HDM2 half-life was quantitated as for p53.

A model for regulated MDM2 destabilization in DNA-damaged cells. (A) In unstressed cells, growth and survival factors activate MDM2 transcription, maintaining basal levels of MDM2 RNA and protein. The resultant MDM2 protein keeps p53 inactive by (1) preventing p53 transactivation by blocking its access to transcriptional co-activators and (2) ubiquitinating the p53 C-terminus leading to its proteasome-mediated degradation. MDM2 auto-ubiquitination keeps its steady-state protein levels low. (B) In DNA-damaged cells, stress-dependent kinases modify MDM2, resulting in its accelerated auto-ubiquitination and proteasome-mediated degradation. MDM2 destabilization frees modified p53 to initiate transcription of high levels of MDM2, as well as genes involved in cell cycle arrest and apoptosis. However, persistent DNA-damage signaling prevents the newly transcribed and translated MDM2 from antagonizing p53 by inducing the rapid degradation of MDM2 protein. This process results in high levels of stable and active p53 that is maintained concurrently with high levels of unstable and ineffective MDM2. (C) Once the DNA damage is repaired, damage kinase signaling abates and re-stabilized MDM2 can bind and inactivate the p53 that had previously accumulated to high levels. Thus, DNA-damage-induced p53-dependent transactivation of MDM2 might provide the cell with a pool of MDM2 that can eliminate active p53 at later times when the stress is alleviated.