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Science 336 (6087): 1440-1444

Copyright © 2012 by the American Association for the Advancement of Science

p53 Dynamics Control Cell Fate

Jeremy E. Purvis, Kyle W. Karhohs, Caroline Mock, Eric Batchelor*, Alexander Loewer{dagger}, and Galit Lahav{ddagger}

Department of Systems Biology, Harvard Medical School, Boston, MA 02115, USA.


Figure 1
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Fig. 1. Perturbation of p53 dynamics. (A) p53 mediates the response to multiple cellular stresses and evokes diverse cellular outcomes. (B) {gamma}-irradiation leads to p53 pulses and cell cycle arrest; UV radiation induces a single prolonged pulse and leads to apoptosis. (C) p53’s natural pulses were perturbed to produce a sustained response with equal amplitude, a. (D) A diagram capturing the main species and parameters in the mathematical model of p53 dynamics after DNA damage (26). This model was used to predict the optimal sequence of Nutlin-3 additions needed to generate a sustained p53 response after {gamma}-irradiation (supplementary materials). (E) p53 dynamics under (left) naturally pulsed or (right) pharmacologically sustained conditions. The sequence of Nutlin-3 treatments is denoted by differently colored bars. Pulses in immunoblots appear as damped oscillations because of the asynchronous responses of single cells. Representative single-cell traces show average nuclear p53-Venus intensities that were normalized to the median value and zeroed to the minimum value. Sequential Nutlin-3 treatment did not alter the amplitude of p53 (fig. S3).

 

Figure 2
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Fig. 2. Pulsed and sustained p53 signaling activate different sets of target genes. Expression of p53 target genes was measured under pulsed or sustained conditions after {gamma}-irradiation. Genes are grouped according to function: (A) cell cycle arrest and DNA repair, (B) control of p53 levels, (C) apoptosis, and (D) senescence. For reference, p53 protein levels are shown in the background as light blue (pulsed) or red (sustained) bars. p53 levels are normalized to the peak (t = 2 hours) p53 concentration. A base-2 logarithmic scale is used for CDKN1A, GADD45A, and MDM2. Data are mean ± SD. Significance of correlation between target genes and p53 protein levels under pulsed conditions is reported in table S3.

 

Figure 3
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Fig. 3. Pulsed and sustained p53 signaling lead to different cell fates. (A) Cells were subjected to pulsed (top) or sustained (bottom) conditions for 3 days and then stained for β-gal activity 1 day after recovery. Blue color and flattened morphology are indicative of senescence. (B) Percentage of β-gal-positive cells under pulsed (P) or sustained (S) p53 signaling at various {gamma}-irradiation doses. n ≥ 100 cells per condition per experiment. *P < 0.05, **P < 0.01. (C) Typical images of single cells that were recovered after 3 days of pulsed (top) or sustained (bottom) p53 signaling at 5 Gy {gamma}-irradiation. White boxes are drawn to show the fate of an individual cell. (D) Number of cell divisions for single cells after recovery from pulsed or sustained p53 signaling at 5 Gy. (E) (Left) Percentage of cells that did not divide under resting conditions or sequential Nutlin-3 treatment alone (no {gamma}-irradiation). (Right) Percentage of nondividing cells under pulsed (P) or sustained (S) p53 signaling. (F) Fold change in expression of CDKN1A, PML, and YPEL3 after 24, 48, and 72 hours under pulsed (blue) or sustained (red) p53 signaling (10 Gy {gamma}-irradiation). Expression levels were normalized to ACTB. Data are mean ± SD.

 

Figure 4
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Fig. 4. p53 dynamics, and not its cumulative level, control cell fate. (A) (Inset) Time points for which pulsed and sustained p53 signaling show equivalent cumulative p53 levels [Formula] lie along the gray line. Gene expression under pulsed (blue dots) or sustained (red dots) p53 signaling are plotted as a function of cumulative p53 by using the data presented in Fig. 1E. The time integral of p53 protein levels was computed by using trapezoidal integration of p53 levels over time. Gene expression was normalized to ACTB. The last time point in sustained conditions (24 hours) was omitted because there is no comparable data point under pulsed conditions. (B) p53 dynamics were recorded by means of live-cell microscopy under pulsed and sustained conditions. (C and D) Representative single-cell traces of p53 levels under (blue) pulsed or (red) sustained conditions. Time-lapse imaging was terminated by fixing cells 21 hours after irradiation (pulsed conditions, t1) or 12 hours after irradiation (sustained conditions, t2) and probing for expression of CDKN1A or PML by means of FISH. (E) CDKN1A and (F) PML expression versus cumulative p53 in individual cells. a.u., arbitrary units; r.f.u., relative fluorescence units (supplementary materials). (G) Model for p53 dynamics controlling cell fate. Transient damage encountered under low-radiation dose or physiological conditions is repaired quickly and generates a small number of p53 pulses, allowing the cell to continue dividing. Persistent damage—whether from a large number of initial DNA lesions or a small number of irreparable breaks—generates repeated p53 pulses that ultimately trigger cellular senescence. t1 and t2 represent time points in which the cumulative level of p53 is equal between pulsed and sustained conditions. However, the probability of entering senescence differs between these two types of dynamics. Pulsed p53 allows more time for recovery from DNA damage, whereas sustained p53 accelerates this process.

 


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