Kinetics of the UV-induced DNA damage response in relation to cell cycle phase. Correlation with DNA replication

Abstract

It has been reported that exposure to UV light triggers DNA damage response (DDR) seen as induction of gammaH2AX not only in S- but also in G(1)-phase cells. In the present study, in addition to gammaH2AX, we assessed other markers of DDR, namely phosphorylation of ATM on Ser1981, of ATM/ATR substrate on Ser/Thr at SQ/TQ cluster domains and of the tumor suppressor p53 on Ser15, in human pulmonary carcinoma A549 cells irradiated with 50 J/m(2) of UV-B. Phosphorylation of these proteins detected with phospho-specific Abs and measured by laser scanning cytometry in relation the cell cycle phase was found to be selective to S-phase cells. The kinetics of phosphorylation of ATM was strikingly similar to that of ATM/ATR substrate, peaking at 30 min after UV irradiation and followed by rapid dephosphorylation. The peak of H2AX phosphorylation was seen at 2 h and the peak of p53 phosphorylation at 4 h after exposure to UV light. Local high spatial density of these phospho-proteins reported by intensity of maximal pixel of immunofluorescence in the DDR nuclear foci was distinctly more pronounced in the early compared to late portion of S-phase. Exposure of cells to UV following 1 h pulse-labeling of their DNA with 5-ethynyl-2'deoxyuridine (EdU) made it possible to correlate the extent of DNA replication during the pulse with the extent of the UV-induced H2AX phosphorylation within the same cells. This correlation was very strong (R(2) = 0.98) and the cells that did not incorporate EdU showed no evidence of H2AX phosphorylation. The data are consistent with the mechanism in which stalling of DNA replication forks upon collision with the primary UV-induced DNA lesions and likely formation of double-strand DNA breaks triggers DDR. The prior reports (including our own) on induction of gammaH2AX in G(1) cells by UV may have erroneously identified cells initiating DNA replication following UV exposure as G(1) cells due to the fact that their DNA content did not significantly differ from that of G(1) cells that had not initiated DNA replication.

Fig. 1. Activation of ATM and phosphorylation of ATM/ATR protein substrates after irradiation of A549 cells with 50 J/m² of UV-B light

The cells growing on microscope slides in slide-chambers were exposed to 50 J/m² of UV light then transferred into culture and the cultures terminated at different time points, as shown in the top panels. The data are presented as ATM-Ser1981^P or ATM/ATR substrate^P vs DNA content bivariate distributions. The insets in the bottom panels show the DNA content frequency histograms of cells from the culture at 0 time and 6 h after irradiation. See text for the details defining subpopulations of cells (a, b, c, d) marked by the dashed-line outlines. .

Fig. 2. Phosphorylation of H2AX on Ser139 and p53 on Ser15 after exposure of A549 cells to 50 J/m² of UV-B light

The cells growing on slides were irradiated with UV light, as described in the legend to Fig 1, and then were cultured for different time intervals as marked. As in Fig. 1, subpopulations a, b, c and d were identified.

Fig. 3. Different patterns of ATM-Ser1981^P and ATM/ATR substrate^P expression across the S-phase cells depending upon whether IF is expressed as intensity of fluorescence integrated over the nucleus (Int) or as maximal pixel of IF in the nucleus (Max pix)

The dashed horizontal or skewed lines are drawn to show the modal values of IF of S-phase cells. Note the increased intensity of IF when presented as maximal pixel in early S cells (eS) as compared with the late S (lS) cells, and no differences between eS and lS in the intensity of the integrated IF.

Fig. 4. Kinetics of ATM, ATR/ATM substrate, H2AX and p53 phosphorylation after exposure of A549 cells to 50 J/m² of UV-B

The data show mean values of IF of the detected phosphorylated proteins of the S-phase cells (gated between DI = 1.2 and DI = 1.8) measured at different time intervals after irradiation with UV.

Fig. 5. Correlation between DNA replication and the UV-induced H2AX phosphorylation in A549 cells

Exponentially growing cells were pulse-labeled with EdU for 60 min then were exposed to UV light and subsequently incubated for 30 min before culture termination. The incorporated EdU was detected by the “click-iT™” methodology using Alexa Fluor 488 azide (green fluorescence), γH2AX was detected using Alexa Fluor 633 secondary Ab (far red fluorescence), DNA was counterstained with DAPI. Using paint-a-gate analysis, the EdU incorporating cells (above the skewed dashed line, panel A) were colored red. As shown in panel B the red colored cells demonstrate expression of γH2AX. Panel C shows the bivariate analysis of γH2AX expression vs EdU incorporation. A strong correlation (R²=0.98) between the extent of H2AX phosphorylation (intensity of γH2AX IF) and the level of EdU incorporation is seen among the EdU incorporating cells outlined with the oval dashed line (the regression plot is shown by the solid line). The subpopulation of cells with increased expression of γH2AX but not incorporating EdU (Sel) was secondarily gated and electronically painted green. These cells were characterized by high intensity of maximal pixel of DAPI fluorescence revealing their highly condensed chromatin. Imaging of these cells by iCys (“Compu-sort”) revealed that they were pre-mitotic or mitotic (M) cells (panels D,E).

Fig. 6. Suppression of EdU incorporation by exposure of cells to UV light

The two left panels present control cells untreated with UV and incubated with EdU for 60 min. The two right panels present the cells that were treated with 50 J/m² of UV, then transferred to culture and incubated with EdU for 1 h. The subpopulation of cells entering S phase (initiating DNA replication) during incubation with EdU is outlined (b).