Journal
CYTOMETRY PART A
Volume 77A, Issue 3, Pages 285-293Publisher
WILEY
DOI: 10.1002/cyto.a.20839
Keywords
ATM activation; ATR activation; gammaH2AX; p53 phosphorylation; checkpoint; apoptosis; laser scanning cytometry; dephosphorylation; G(1) phase
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Funding
- NCI NIH HHS [CA 28704, R01 CA028704-31, R01 CA028704-30, R01 CA028704] Funding Source: Medline
- NCRR NIH HHS [S10 RR026548, S10 RR026548-01] Funding Source: Medline
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It has been reported that exposure to UV light triggers DNA damage response (DDR) seen as induction of gamma H2AX not only in S- but also in G(1)-phase cells. In the present study, in addition to gamma H2AX, 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 gamma H2AX 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. (C) 2009 International Society for Advancement of Cytometry
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