First published online 25 July 2006
doi: 10.1242/jcs.03068
Journal of Cell Science 119, 3284-3295 (2006)
Published by The Company of Biologists 2006
Promyelocytic leukemia nuclear bodies are predetermined processing sites for damaged DNA
Stig Ove Bøe1,2,*,
Marte Haave1,2,
Åsne Jul-Larsen1,2,
Amra Grudic1,2,
Rolf Bjerkvig2,3 and
Per Eystein Lønning1
1 Section of Oncology, Department of Medicine, Haukeland University Hospital, Bergen, Norway
2 Section of Anatomy and Cell Biology, Department of Biomedicine, University of Bergen, Jonas Lies vei 91, N-5009, Bergen, Norway
3 NorLux, Neuro-Oncology, Centre Recherche Public Santé, Luxembourg
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Fig. 1. Formation of ssDNA foci in GM-847 cells in response to UV-irradiation. GM-847 cells metabolically labeled for 24 hours with BrdU were subjected to 10 J/m2 UV-irradiation and subsequently incubated for 1 or 2 hours in the presence or absence of 4 mM caffeine. (A) Representative immunofluorescence images of non-irradiated cells containing ssDNA-positive APBs and UV-irradiated and caffeine-treated cells containing UV-induced ssDNA foci. (B) Quantitative determination of cells containing ssDNA-foci. For each sample, more than 500 cells were scored. Average of two independent experiments ± standard deviation (s.d.) is shown. (C) Quantitative determination of cells exhibiting co-localization between TRF2 and ssDNA-containing foci. For each sample, 50 cells that contained ssDNA foci were evaluated. Average of two independent experiments ± s.d. is shown. (NI) Non-irradiated.
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Fig. 2. ssDNA focus formation in response to UV-irradiation depends on PML-NB integrity. (A) Western blot of nuclear matrix extracts prepared from GM-847 cells at 48 hours following transfection with PML- or GL2 (control)-specific siRNAs. The band indicated by an asterisk either represents a non-specific background band or a PML-isoform that is resistant to siRNA-mediated depletion. The ß-actin protein is used as a loading control. (B,C) Cells were transfected with the indicated siRNAs on day 1, and on day 2 the growth medium was supplemented with 10 µM BrdU. On day 3 (24 hours following addition of BrdU) cells were UV irradiated (or non-irradiated) and subsequently grown in the presence or absence of 4 mM caffeine for 1 or 2 hours before fixation. (B) Representative immunofluorescence images of siRNA-transfected GM-847 cells at 2 hours following irradiation. (C) Quantitative determination of cells containing ssDNA foci. For each sample, more than 500 cells were scored. The average of two independent experiments ± s.d. is shown. (D) GM-847 cells were transfected with the plasmid pYFP-PML + carrier plasmid (see Materials and Methods) and immediately after subjected to BrdU-labeling for 24 hours. Cells were then treated with UV light (or left non-irradiated) followed by incubation in the presence of caffeine for 90 minutes prior to fixation. The YFP signal and the immunofluorescently labeled BrdU are shown. (E) Quantitative determination of YFP-positive cells that contain ssDNA foci following UV-irradiation and caffeine treatment (same experiment as D). For each sample, 100 YFP-positive cells were scored. The average of two independent experiments ± s.d. is shown. (NI) Non-irradiated.
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Fig. 3. ssDNA focus formation within PML-NBs is not associated with apoptotic DNA fragmentation. (A) Cells were treated with BrdU for 24 hours and subsequently irradiated with 10 J/m2 UV light or non-irradiated. Cells were then incubated in the presence or absence of caffeine for 1 or 2 hours before analysis by immunofluorescence (upper panels) or pulse field gel electrophoresis (lower panels). Bars in upper panels represent quantification of cells containing ssDNA foci. For each sample, more than 400 cells were evaluated. The average of two independent experiments ± s.d. is shown. (B) GM-847 cells were incubated in the presence of BrdU for 24 hours and then treated with the drugs indicated for 3 hours. Following treatment, cells were analyzed as in panel A by immunofluorescence labeling (upper panel) and pulse field gel electrophoresis (lower panel).
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Fig. 4. Inhibition of PARP-1 stimulates UV-induced ssDNA focus formation. (A) GM-847 cells seeded on coverslips were transfected with PML-specific siRNAs or control siRNAs (GL2) as indicated. BrdU was added to the culture medium at 24 hours after transfection. At 48 hours post-transfection cells were treated with 10 J/m2 UV light and subsequently incubated in the presence or absence of 0.5 mM NU1025 for 1 or 2 hours before fixation and immunofluorescence analysis. Data represent the percentage of cells containing ssDNA foci. For each sample more than 300 cells were evaluated. The data represent the average of two independent experiments ± s.d. (B) Western blot of extracts prepared from GM-847 cells at 72 hours following transfection with siRNAs specific for PARP-1 and GL2 (control). The MCM2 protein is used as a loading control. (C) GM-847 cells were transfected with the siRNAs indicated. BrdU was added to the growth medium at 48 hours following transfection. At 24 hours following addition of BrdU, cells were irradiated with 10 J/m2 UV light or non-irradiated and subsequently incubated in normal growth medium for 1 or 2 hours. (NI) Non-irradiated.
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Fig. 5. UV-induced ssDNA focus formation is stimulated during S-phase. (A) GM-847 cells treated with nocodazole for 14 hours were subjected to mitotic shake-off and subsequently seeded on coverslips. At the indicated time-points, cells were pulsed with BrdU, and the number of cells in S-phase were determined by immunofluorescence labeling using an anti-BrdU antibody. For each sample more than 400 cells were scored. The data represent the average of two independent experiments ± s.d. (B) GM-847 cells were treated with BrdU for 24 hours and subsequently subjected to nocodazole treatment and mitotic shake-off in parallel with the experiment described in A. At the indicated time-points following mitotic shake-off, cells were UV-irradiated with 10 J/m2, and subsequently incubated in the presence of 4 mM caffeine for 2 hours. The number of cells containing PML-associated ssDNA foci were determined by immunofluorescence labeling. For each sample more than 400 cells were scored. The data represent the average of two independent experiments ± s.d. (C) Representative images of UV-induced PML-associated ssDNA foci in G1 and S-phase cells formed by UV-irradation at 5 and 12 hours post mitotic shake-off, respectively.
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Fig. 6. ssDNA focus formation is stimulated by S-phase progression. (A) GM-847 cells were transfected with the indicated siRNAs, and BrdU was immediately added to the growth medium. At 24 hours following transfection, BrdU was removed from the cells and mimosine or HU were added. 24 hours thereafter, drugs were removed and the cells were incubated in normal growth medium until fixation at the indicated time-points. (B) ssDNA focus formation following release from mimosine block. The upper panel shows laser scanning flow cytometry analysis of TO-PRO-3-labeled cells. The lower panel shows quantitative determination of cells containing BrdU-labeled ssDNA foci. Bars represent the percentage of cells containing ssDNA foci. For each sample, more than 200 cells were evaluated. Data represent the average of two independent experiments ± s.d. (C) ssDNA focus formation following release from HU block. The same experiment as in B, except HU was used instead of mimosine. `C' indicates samples where cells have not been treated with mimosine or HU.
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Fig. 7. Effect of PML depletion on cell cycle progression in the presence of DNA damage caused by etoposide. GM-847 cells were transfected with control siRNAs (GL2) or PML-specific siRNAs, and at 24 hours following transfection the culture medium was supplemented with 10 µM BrdU. At 48 hours post-transfection the BrdU-containing medium was removed and cells were incubated for another 24 hours in medium containing the indicated concentrations of etoposide. (A) Quantitative determination of cells containing ssDNA foci within PML-NBs. For each sample, more than 500 immunofluorescence labeled cells were evaluated. Bars represent the average of two independent experiments ± s.d. (B) Western blot analysis of soluble cell extracts using an antibody specific for histone H3 phosphorylated on Ser10. The MCM2 protein is used as loading control. (C) Analysis of To-Pro-3-stained cells by laser scanning cytometry.
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Fig. 8. Effect of PML depletion on UV-induced apoptosis. GM-847 cells were transfected with PML-specific siRNAs or control siRNAs (GL2) and subjected to the indicated doses of UV irradiation on day 3 following transfection. Subsequent to irradiation, cells were incubated in the absence or in the presence of 4 mM caffeine for 2 hours prior to analysis. (A,B) Cells were fixed, stained with Hoechst and evaluated by immunofluorescence microscopy for the presence or absence of condensed chromatin. For each sample, more than 500 cells were evaluated. The data represents the mean ± s.d. of two independent experiments. (C,D) Analysis of apoptotic DNA fragmentation by pulse field gel electrophoresis. The 50-300 kb apoptotic DNA fragments are indicated.
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© The Company of Biologists Ltd 2006
