QUICK SEARCH:   [advanced] Author: Keyword(s):
Home     Help     Feedback     Subscriptions     Archive     Search     Table of Contents

First published online September 29, 2004
doi: 10.1242/10.1242/jcs.01374

This Article Summary Full Text Full Text (PDF) Supplemental Material Alert me when this article is cited Alert me if a correction is posted Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Szüts, D. Articles by Krude, T. Search for Related Content PubMed PubMed Citation Articles by Szüts, D. Articles by Krude, T.

Cell cycle arrest at the initiation step of human chromosomal DNA replication causes DNA damage

Department of Zoology, University of Cambridge, Downing Street, Cambridge CB2 3EJ, UK

View larger version (19K): [in a new window]   Fig. 1. Cell cycle arrest at the G1-S phase boundary caused by iron chelators. (A) Flow cytometry analysis of propidium iodide stained nuclei, isolated from HeLa cells that were treated for 24 hours with the iron chelators mimosine, ciclopirox olamine or 2,2'-bipyridyl, and, as control, with the replication-elongation inhibitor aphidicolin, at the indicated concentrations. The positions of unreplicated (2n) and fully replicated DNA content (4n) are indicated. (B) In vitro replication reactions of nuclei, isolated from cells treated with the iron chelators and aphidicolin as in A. Nuclei were incubated in an elongation buffer (`buffer', light grey bars) or in the same buffer supplemented with 100 µg HeLa cell cytosolic extract (cytosol, dark grey bars). Percentages of nuclei that incorporated labelled nucleotides were determined. The mean of 3-4 experiments and the standard deviations are shown.

View larger version (43K): [in a new window]   Fig. 2. The cell cycle arrest caused by iron chelation is reversible. (A) HeLa cells were kept in 500 µM mimosine for 24 hours. Then, either mimosine was removed by replacing the medium (mimosine release, rel) or 1 mM FeSO4 (Fe) was added. Nuclei were prepared and their DNA content was analysed by flow cytometry at the indicated time points. The `0 h' control indicates a 24-hour treatment with mimosine only. (B) Proportion of cells in the G1, S or G2-M phases of the cell cycle at the indicated time points after release from mimosine-induced cell cycle arrest, as determined from the flow cytometry histograms shown in A. (C) In vitro replication assay of nuclei from the 0, 1, 2 and 4-hour samples from A. Nuclei were incubated in elongation buffer and the percentage of nuclei that incorporated labelled nucleotides is shown for each time point.

View larger version (106K): [in a new window]   Fig. 3. Treatment with iron chelators causes DNA breaks. (A) EJ30 cells were treated with the indicated compounds for 24 hours, fixed, treated with RNase A and stained with propidium iodide for DNA (red) and with antibodies for -H2AX (green). Merged images are shown. (B) HeLa cells were subjected to treatments exactly as in A and the generated DNA breaks were analysed by alkaline-agarosegel electrophoresis. Lanes 1 and 7, untreated control; 2, thymidine; 3, aphidicolin; 4, mimosine; 5, ciclopirox olamine; 6, bipyridyl; 8, etoposide; 9, 1 µg/ml Bleocin; 10, 10 µg/ml Bleocin. The marker (M) is a 1 kb ladder (Roche). (C) Samples 1-4 from B were subjected to neutral pulsed-field-gel electrophoresis to assay for the presence of DSBs. The markers cover the 0.1-200 kb (M1) or 50-1000 kb (M2) size-range.

View larger version (58K): [in a new window]   Fig. 6. Mimosine-induced DNA damage is cell cycle dependent. (A) Quiescent EJ30 cells were incubated in 500 µM mimosine for 24 hours, fixed, treated with RNase A, and stained for DNA (red) and for -H2AX (green). (B) Proliferating EJ30 cells were incubated for 24 hours and stained as in A. (C) EJ30 cells were arrested in mitosis by a 24-hour nocodazole treatment, released by replacing the medium, and then subjected to a treatment of 500 µM mimosine or 5 µg/ml aphidicolin in intervals of 4-8, 8-12 and 12-16 hours post release. The percentage of cells showing focal -H2AX staining were determined by immunofluorescence microscopy. (D) Flow-cytometry analysis of the DNA content of untreated control cells at the indicated time points after release from nocodazole as used in C. The percentages of cells that incorporated BrdU in the 15 minutes before preparation are shown in each panel.

View larger version (57K): [in a new window]   Fig. 4. Activation of DNA damage response pathways. EJ30 cells were grown on coverslips and were either used as control (untreated) or treated with 500 µM mimosine for 24 hours. After fixation, the cells were stained with antibodies against (A) -H2AX (green) and RPA (red), (B) RPA34 (green) and ATR (red) and (C) -H2AX (green) and phosphorylated CHK1-S317-P (red). Representative nuclei are shown in A and B. (C) To illustrate the correlation between the intensity of -H2AX staining and the level of CHK1-P, a low magnification field of nuclei is shown. Merged RGB images are shown in the right hand panels.

View larger version (96K): [in a new window]   Fig. 7. DNA replication proceeds in the presence of DNA breaks upon release from mimosine. (A) Control EJ30 cells and cells treated with 500 µM mimosine for 24 hours were stained for -H2AX (green) and DNA (red). (B) Cells, stained as in A, 4 or 24 hours after the removal of mimosine. (C) Cells, stained as in A, 4 or 24 hours after the addition of 1 mM FeSO4, which was added 24 hours after the addition of 500 µM mimosine. (D) Graph, showing the percentage of cells with strong focal -H2AX staining, as seen in B-C, at the indicated time points after cells had been released from arrest by either mimosine removal (squares) or iron addition (circles). (E) Western blot, showing the phosphorylation of the CHK1, CHK2 and H2AX proteins in whole cell extracts, prepared at the indicated time points after release from a mimosine arrest; the arrow indicates the specific -H2AX band. (F) HeLa cells, subjected to identical treatments as in A-C. The presence of DNA breaks was analysed by alkaline gel electrophoresis as in Fig. 3B. (G) EJ30 cells were treated with 500 µM mimosine for 24 hours and assayed for their competence to initiate DNA replication in the presence of DNA damage in vivo (top panels) or in vitro (bottom panels). In vivo, cells were released into fresh medium containing BrdU for 1 hour, fixed and stained for -H2AX (using a rabbit polyclonal antibody, green) and BrdU (red). For the in vitro test, nuclei of mimosine-treated cells were incubated for 5 minutes in a replication assay mix containing cytosolic extract and digoxigenin-dUTP (Roche), fixed and stained for -H2AX (green) and digoxigenin (red). Representative nuclei of the class that stains for both -H2AX and replication markers are shown. Merged RGB images are shown in the right hand panels.

View larger version (51K): [in a new window]   Fig. 5. The iron depletion-induced cell cycle arrest is independent of checkpoint activation. (A) EJ30 cells were subjected to 24-hour treatments with mimosine or aphidicolin in the absence or presence of 25 µM wortmannin, and stained for DNA (red) and -H2AX (green). (B) Representative cell nuclei from these experiments, stained for RPA. (C) Flow cytometry profiles of HeLa cells treated with mimosine for 28 hours (top); with mimosine only for 24 hours, followed by mimosine plus wortmannin for 4 hours (middle); or with mimosine only for 24 hours, followed by mimosine plus caffeine for 4 hours (bottom). (D) Nuclei from cells subjected to 24-hour treatments with mimosine (mim) or wortmannin (wort) or both (mim/wort) as in A were analysed by in vitro replication reactions. Nuclei were incubated in an elongation buffer (buffer) or in the same buffer supplemented with 100 µg HeLa cell cytosolic extract (cytosol). Percentages of nuclei incorporating labelled nucleotides are shown. (E) Nuclei from cells subjected to 24-hour treatments with caffeine (caff), mimosine (mim) or both (mim/caff) were analysed by in vitro replication reactions as in D.