DNA damage checkpoint responses in the S phase of synchronized diploid human fibroblasts

Abstract

We investigated the hypothesis that the strength of the activation of the intra-S DNA damage checkpoint varies within the S phase. Synchronized diploid human fibroblasts were exposed to either 0 or 2.5 J m(-2) UVC in early, mid- and late-S phase. The endpoints measured were the following: (1) radio-resistant DNA synthesis (RDS), (2) induction of Chk1 phosphorylation, (3) initiation of new replicons and (4) length of replication tracks synthesized after irradiation. RDS analysis showed that global DNA synthesis was inhibited by approximately the same extent (30 ± 12%), regardless of when during S phase the fibroblasts were exposed to UVC. Western blot analysis revealed that the UVC-induced phosphorylation of checkpoint kinase 1 (Chk1) on serine 345 was high in early and mid S but 10-fold lower in late S. DNA fiber immunostaining studies indicated that the replication fork displacement rate decreased in irradiated cells at the three time points examined; however, replicon initiation was inhibited strongly in early and mid S, but this response was attenuated in late S. These results suggest that the intra-S checkpoint activated by UVC-induced DNA damage is not as robust toward the end of S phase in its inhibition of the latest firing origins in human fibroblasts.

Figure 1

Aphidicolin treatment of cells released from confluence arrest improves the synchronized progression of human fibroblasts through the S phase. (A) Schematic of the synchronization protocol (not drawn to scale). (B) Bivariate flow cytometric analysis of NHF1-hTERT labeled with BrdU for 30 min starting 45 min, 3 h and 5 h into S; the DNA content was measured by propidium iodide staining. The percentage of cells in S at these time points was 70, 78 and 49%, respectively.

Figure 2

UVC inhibits DNA synthesis in cells synchronized to early, mid- or late-S phase. NHF1-hTERT cells were sham treated or irradiated with 2.5 J m⁻² UVC at the indicated times and pulsed for 30 min with ³H-thymidine. The incorporation of ³H-thymidine into acid-insoluble macromolecules was normalized to absorbance at 260 nm (see Materials and Methods for details). Next, the relative CPM/A260 values were determined by dividing the normalized ³H-thymidine incorporation in each sample by the average CPM/A260 in the sham group within an experiment. The illustrated data represent averages of these calculated values (n = 4); the error bars indicate the standard error of the mean. Irradiation inhibited the overall DNA synthesis rate in early (P = 0.0006), mid (P < 0.0001) and late-S phase (P < 0.0001); the UVC-induced inhibition was 23 ± 15% in early S, 28 ± 8% in mid S and 39 ± 8% in late-S phase; no significant differences in the magnitude of this inhibition was found between early and mid S (P = 0.64), while significant differences were found between early and late S (P = 0.0013) and between mid and late S (P = 0.0055).

Figure 3

Cells late in S do not inhibit origin initiation after UVC exposure as efficiently as in early and mid S phase. (A) Left panel: Schematic illustration of the labeling patterns of active replication units before (IdU, red) and after (CldU, green) irradiation with 0 or 2.5 J m⁻² of UVC. Right panel: Examples of replication units labeled through a 10-min pulse with IdU followed by a 20-min pulse with CldU; photomicrographs and schematic drawings of the corresponding fibers. (B) Percent of CldU-only replication tracks (new initiation) in NHF1-hTERT and NHF10-hTERT after treatment with 0 or 2.5 J m⁻² UVC at 45 min, 3 h or 5 h into S phase. UVC exposure in NHF1-hTERT was associated with a strong inhibition of replicon initiation at 45 min (P = 0.0031) and 3 h (P = 0.0096), while an attenuated response was observed at 5 h (P = 0.0855); see Table1. The error bars represent the standard errors of the mean with n = 4. Results of a single experiment with NHF10-hTERT agreed well with those obtained with NHF1-hTERT.

Figure 4

The level of Chk1-P increases when S phase cells are irradiated, but this response is attenuated in late S. (A) Immunoblot for total and phosphorylated Chk1 from a representative synchronization experiment. Equal amounts of protein from NHF1-hTERT that were sham treated or irradiated with UVC (2.5 J m⁻²) at 45 min, 3 h or 5 h into S and incubated for 30 min in reserved medium were separated by SDS-PAGE; the transferred proteins were immunostained for total Chk1 or Chk1-P (Ser³⁴⁵). (B) Normalized Chk1-P/total Chk1 values (average ± standard error of the mean) from six independent experiments (see Materials and Methods for details). UVC induced the phosphorylation of Chk1 when the cells were irradiated at 45 min (P = 0.0004) and 3 h (P = 0.002) but not when the cells were irradiated at 5 h (P = 0.44). There was no significant difference between the UVC induction of Chk1-P at 45 min and 3 h (P = 0.97), while the response at 5 h significantly differed compared to that at 3 h (P = 0.002).

Figure 5

UVC-induced Chk1 phosphorylation is attenuated in late S in cells synchronized only by release from confluence arrest. (A) Schematic drawing of the synchronization protocol. (B) Bivariate flow cytometric analysis of NHF1-hTERT labeled with BrdU for 60 min starting 15, 18, 21 or 24 h after release from confluence arrest. The percentage of cells in S at 15, 18, 21 or 24 h after release from confluence arrest was 44, 41, 23 and 16%, respectively. (C) Equal amounts of protein from NHF1-hTERT cells that were sham treated or irradiated with UVC (2.5 J m⁻²) at 15, 18, 21 or 24 h were separated by SDS-PAGE; the transferred proteins were immunostained for Chk1 and Chk1-P (Ser³⁴⁵) kinase. The histogram shows the Chk1-P signal normalized to the total Chk1 at each time point for the sham-treated and irradiated cells. (D) The experiment described in (A–C) was repeated in NHF10-hTERT; the histogram shows the Chk1-P signal normalized to total Chk1 at each time point for sham-treated and irradiated cells. The percentage of cells in S at these time points was 24, 27, 17 and 8%, respectively.