Research ArticleCell Biology

p53 dynamics in response to DNA damage vary across cell lines and are shaped by efficiency of DNA repair and activity of the kinase ATM

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Science Signaling  25 Apr 2017:
Vol. 10, Issue 476, eaah6671
DOI: 10.1126/scisignal.aah6671
  • Fig. 1 p53 abundance and the initial response to DNA damage are comparable across cancer cell lines.

    (A) Twelve cell lines were stained for p53 before or 2 hours after treatment with the DNA-damaging agent NCS (100 ng/ml). Scale bar, 50 μm. “M” marks the five melanoma lines. (B) Histograms of single-cell straining intensity of p53 before (blue) or after (red) treatment with NCS. Black lines indicate the medians of the distributions. Cells are ordered by their median p53 abundance after NCS. AU, arbitrary units. (C and D) A549 cells were stained for p53 at 2 hours (C) or 8 hours (D) after a log2 titration series of NCS. Red lines indicate the median of the distribution. Variation in p53 abundance across doses was assessed by unpaired t tests: P > 0.05 at 2 hours; P < 0.05 at 8 hours. (E and F) Each cell line was stained for p53 at 2 hours (E) or 8 hours (F) after varying doses of NCS. Heat maps represent p53 abundance. Each cell line was internally normalized (Norm.) between 0 and 1. Data in (B) to (F) are representative of two independent experiments (n > 500 cells for each cell line).

  • Fig. 2 p53 shows different temporal patterns across cell lines.

    (A and B) Abundance of p53 and MDM2 was quantified by immunofluorescence and is plotted as boxplots showing the change (Δ) in protein abundance between the 0- and 2-hour time points or between the 2- and 8-hour time points after NCS treatment (100 ng/ml). Dots represent each of the 12 cell lines, highlighting MCF7 (green), A549 (red), and HCT116 (blue). (C) Abundance of p53 quantified by immunofluorescence at the indicated time points after NCS treatment. Histograms from the three cell lines are shown. Data in (A) to (C) are representative of two independent experiments (n > 500 cells for each cell line).

  • Fig. 3 Live-cell tracking of p53 over time reveals cell line–specific dynamics and dose dependency.

    (A) Images of MCF7 and A549 cell lines expressing p53-YFP over 18 hours after 1- or 6-gray (Gy) IR exposure. Scale bar, 50 μm. (B) Heat maps of p53-YFP abundance in MCF7 and A549 cells after exposure to various doses of IR. Each row represents a single cell. (C to F) Twelve cell lines were constructed to express p53-YFP and imaged after exposure to IR: 1 Gy, blue line; 2 Gy, red line; 4 Gy, yellow line; 6 Gy, purple line; 8 Gy, green line [or boxplot in (F)]. Data are from n > 50 cells for each condition (3825 cells in the full data set; pooled from two to three experiments for each line). “M” indicates melanoma cell lines. Measured for each dose in each cell line were the fraction of cells that have divided at a given time (C), p53-YFP abundance (bold colored lines, averages; gray lines, single-cell traces) (D), average autocorrelations of p53 trajectories (E), and the FWHMs of the first p53 pulse (boxplots show the distribution over single cells; asterisks indicate cell lines where the FWHM is dose-dependent; P < 0.05, t test) (F). (G) A measure of periodicity (maximum of autocorrelation function minus the minimum of autocorrelation function in the first 5 hours; see inset) was calculated for each cell line and condition.

  • Fig. 4 A kinase inhibitor screen in live cells identified compounds that alter p53 dynamics after DNA damage.

    (A) Screen workflow in which U2OS cells expressing p53-YFP were incubated for 2 hours with kinase inhibitors and then exposed to NCS (100 ng/ml), followed by live-cell imaging of p53-YFP abundance over 24 hours. (B) Effect of pretreatment with various signaling molecule inhibitors on NCS-induced p53-YFP dynamics in U2OS cells. In the control graph [dimethyl sulfoxide (DMSO); far left], the bold blue line is the average trace, and the gray lines are single-cell traces. Each graph then shows the control trace as a reference (bold blue trace) alongside colored traces representing the average trajectory of p53-YFP abundance in cells exposed to specific compounds within each class of drug [each of eight CDK inhibitors (CDKi), two FAK inhibitors (FAKi), four mTOR inhibitors (TORi), three ATM inhibitors (ATMi), and four distinct combinations of two PARP (PARPi) and two DNA-PK inhibitors (DNA-PKi)]. Bottom: Heat maps of p53-YFP abundance in single U2OS cells after treatment with NCS (100 ng/ml) and the corresponding drug treatment (color-coded groups correspond to the traces above) (n > 20 cells for each condition). (C and D) Abundance of p53-YFP (C) or the transcriptional reporter MDM2::T2A-GFP (green fluorescent protein) (D) in transfected MCF7 cells exposed to a 3-hour pulse (shaded) of the MDM2 inhibitor (MDM2i) nutlin-3A, the CDK inhibitor flavopiridol, or the FAK inhibitor PF-431396. (E) Abundance of p53-YFP in A549 cells treated with either NCS (100 ng/ml; single-cell traces in gray, average in bold) (top) or the mTOR inhibitor AZD8055 and then NCS (blue, NCS-only trace inserted for reference) (bottom). Data in (C) to (E) are from n > 25 cells, representative of two experiments.

  • Fig. 5 Repair proficiencies vary between cell lines.

    (A) A549 cells expressing p53-YFP were pretreated with DMSO (NT) or olaparib (PARPi; 10 μM), NU7026 (DNA-PKi; 10 μM), or CH99021 (GSK3βi; 10 μM) for 1 hour and then treated with NCS (100 ng/ml). Red lines represent the treated condition (thin lines, single cells; thick line, average), and the blue line represents the control (NT) cells Data are representative of two independent experiments (n > 40 cells). (B) Quantification of γH2AX intensity induced by NCS (100 ng/ml) was measured 6 hours after 10-Gy IR in A549 cells pretreated with DMSO (NT; 1 hour) or the indicated inhibitor (10 μM; 1 hour). Histograms are shown, with a red line indicating the median. Data are representative of three independent experiments (n > 500 cells; P < 0.05, t test). (C) Images of cells from the indicated lines stained for γH2AX before or 0.5 or 24 hours after NCS treatment (100 ng/ml). Scale bar, 25 μm. (D) Histograms of MCF7, A549, and HCT116 cells stained for γH2AX at the indicated time points after NCS treatment (n > 200 cells from two independent experiments). (E) γH2AX in HCT116 cells was quantified 30 min and 8 hours after NCS. (F) DNA damage assessed as γH2AX abundance at 30 min and 8 hours in the indicated cell lines was compared across a range of NCS concentrations (noted in the color scale). Data are means ± SD (n = 7).

  • Fig. 6 ATM signaling varies across cell lines and manipulation of ATM modifies p53 periodicity.

    (A) ATM activity was quantified by immunofluorescence measurements of phosphorylated (p) CHK2 30 min after NCS (100 ng/ml) in the presence of one of the eight doses of ATM inhibitor KU55933. Data are shown as a lineplot, smoothed with a window of three. (B) The median inhibitory concentration (IC50) of each cell line in response to ATM inhibition calculated from (A). (C) Mean p53-YFP abundance measured by immunofluorescence in each cell line treated with ATM inhibitor (2 μM; red) or DMSO (black) and at 2, 5, and 7 hours after NCS (100 ng/ml). Data are representative of three experiments (n > 200 cells). (D and E) Left: p53-YFP abundance in A549 cells exposed to 6-Gy IR (D) or MCF7 cells exposed to 8-Gy IR (E) in the presence (bottom) of DMSO (top) or ATM inhibitor (KU55933, 2 μM) (D) or PPM1D inhibitor (2 μM) (E). Right: Corresponding average autocorrelation curves (n > 50 cells, representative of two independent experiments).

  • Fig. 7 ATM signaling and DNA repair capacity provide a range of p53 dynamical behaviors.

    (A) A computational model of p53 signaling was simulated with varying ATM activities and DNA repair efficiencies, resulting in a range of p53 dynamics capturing the experimental measurements in the different cell lines. (B) The maximum p53 amplitude and periodicity are shown as heat maps for various values of ATM activities and repair efficiencies. The putative “signaling spaces” in which MCF7 or A549 cells may reside are indicated, as are the effects of chemical inhibitors used in this study (right). WT, wild type; PPM1Di, PPM1D inhibitor.

Supplementary Materials

  • www.sciencesignaling.org/cgi/content/full/10/476/eaah6671/DC1

    Text S1. MATLAB simulations.

    Fig. S1. Cell-to-cell variation in p53 abundance is not because of genetic inhomogeneity.

    Fig. S2. Proliferation and induction of p53 target genes after DNA damage across cell lines.

    Fig. S3. Characterization of modulators of p53 dynamics.

    Fig. S4. The fraction of unrepaired DNA breaks is cell line–specific and does not depend on the damage dose.

    Table S1. Summary of chemical screening data.

  • Supplementary Materials for:

    p53 dynamics in response to DNA damage vary across cell lines and are shaped by efficiency of DNA repair and activity of the kinase ATM

    Jacob Stewart-Ornstein and Galit Lahav*

    *Corresponding author. Email: galit{at}hms.harvard.edu

    This PDF file includes:

    • Text S1. MATLAB simulations.
    • Fig. S1. Cell-to-cell variation in p53 abundance is not because of genetic inhomogeneity.
    • Fig. S2. Proliferation and induction of p53 target genes after DNA damage across cell lines.
    • Fig. S3. Characterization of modulators of p53 dynamics.
    • Fig. S4. The fraction of unrepaired DNA breaks is cell line–specific and does not depend on the damage dose.
    • Table S1. Summary of chemical screening data.

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    Citation: J. Stewart-Ornstein, G. Lahav, p53 dynamics in response to DNA damage vary across cell lines and are shaped by efficiency of DNA repair and activity of the kinase ATM. Sci. Signal. 10, eaah6671 (2017).

    © 2017 American Association for the Advancement of Science

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