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First published online 9 September 2008
doi: 10.1242/jcs.027730

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p21 differentially regulates DNA replication and DNA-repair-associated processes after UV irradiation

¹ Cell Cycle and Genomic Stability Laboratory, Fundación Instituto Leloir-CONICET, Universidad de Buenos Aires, Buenos Aires, Argentina
² Laboratory of Molecular and Cellular Therapy, Fundación Instituto Leloir-CONICET, Universidad de Buenos Aires, Buenos Aires, Argentina
³ Department of Biological Sciences, Columbia University, New York, NY 10027, USA

View larger version (52K): [in this window] [in a new window]   Fig. 1. The PCNA-binding domain of p21 is not essential to block cell-cycle progression. (A) U2OS cells were transfected with GFP-PCNA and the indicated p21 plasmids and the cell-cycle profile of the transfected population was determined. (B) U2OS cells were transfected as in A. BrdU (10 µM) was added 30 minutes before fixation and was detected with specific antibodies. Representative images are shown. The percentage of GFP-PCNA-positive cells that incorporated BrdU was determined (bar chart). At least 200 transfected nuclei/sample were counted. Values are the average and error bars are the standard deviation between equivalent samples in two independent experiments. (C) U2OS cells were transfected as in A, and the sub-nuclear distribution of GFP-PCNA was determined. In the case of the empty vector (EV) and 6Mycp21 (CDK–), 60% of the cells showed a diffuse distribution, whereas the remainder showed focal PCNA. The two images shown for EV and 6Mycp21 (CDK–) are representative of each situation (merged panels in supplementary material Fig. S5). The quantification of three independent experiments is reported in Fig. 3B.

View larger version (41K): [in this window] [in a new window]   Fig. 2. Neither the PCNA-binding nor the CDK-binding domain of p21 inhibits early or late steps of NER. (A) U2OS cells transfected with the indicated plasmids were subjected to UV irradiation. Samples were collected at different time points and p21 protein levels were determined using specific antibodies. Actin was used as a loading control. (B) U2OS cells transfected with the indicated plasmids were UV irradiated (80 J/m2) using polycarbonate filters. Thirty minutes later, cells were fixed and immunostained with XPB- and p21-specific antibodies. DAPI staining was used to visualize the nucleus. Quantification is reported in the bar chart. The first column (EV) indicates the percentage of total cells that showed XPB recruitment to the irradiated spots. The other columns represent the percentage of p21-positive cells with XPB relocalization to irradiated spots. In all cases, at least 200 transfected nuclei/sample were counted. Values are the average and error bars are the standard deviation between equivalent samples in two independent experiments. (C) U2OS cells were transfected with the indicated plasmids and GFP-PCNA as a transfection marker. Samples were UV irradiated (20 J/m2) or not (non-irradiated, NI) and BrdU incorporation (100 µM) was performed for 4 hours. Cells were fixed and stained with BrdU-specific antibodies and DAPI. The white outline, generated using confocal software, was used to distinguish nuclear BrdU signal from mitochondrial DNA synthesis. The magnified (Zoom) images show the nuclei indicated by arrowheads. Quantification of the results obtained with all p21 mutants is reported in the bar chart (10 nuclei/sample were quantified). A complete panel showing all the p21 mutants is shown in supplementary material Fig. S3B.

View larger version (30K): [in this window] [in a new window]   Fig. 3. The p21-PCNA interaction impairs the assembly of new GFP-PCNA foci after UV irradiation. (A) U2OS cells transfected with GFP-PCNA and the indicated p21 plasmids were UV irradiated (20 J/m2). Six hours later, cells were fixed and the sub-nuclear distribution of PCNA and p21 determined by confocal microscopy. Non-irradiated (NI) controls obtained in parallel are shown in Fig. 1C. After UV irradiation, PCNA foci were similar to replication PCNA foci (EV, left-hand panel) or much smaller (EV, right-hand panel). When the 6Mycp21 (CDK–) mutant was transfected, the PCNA distribution ranged from diffuse to increasingly collapsed nuclear foci as shown in the set of panels to the right. Merged panels are shown in supplementary material Fig. S5. (B) The percentage of cells with GFP-PCNA foci before and after 6 hours of UV irradiation (20 J/m2) was determined. In all cases, at least 200 transfected nuclei were counted. Values are the average and error bars are the standard deviation between equivalent samples in three independent experiments. The significance of the differences between the NI and UV samples was assessed by Student's t-test for each p21 variant (***P<0.001; ns=P>0.05, not significant). Further statistical comparison was performed by one-way ANOVA with Tukey-Kramer post-test (Table 1). (C) The percentage of cells with GFP-PCNA foci was determined at different time points after UV irradiation (20 J/m2). In all cases, 200 transfected nuclei/sample were counted. Values are the average and error bars are the standard deviation between equivalent samples in two independent experiments.

View larger version (55K): [in this window] [in a new window]   Fig. 4. The PCNA-binding domain of p21 inhibits GFP-pol foci formation after UV irradiation. (A) U2OS cells transfected with GFP-pol and the indicated p21 plasmids were UV irradiated (20 J/m2) when indicated. Six hours later, cells were fixed and the sub-nuclear distribution of pol and p21 determined by confocal microscopy. In control samples, pol foci detected after UV irradiation were of two types: larger and fewer (EV, left-hand panel), or much smaller and greater in number (EV, right-hand panel). When 6Mycp21 (CDK–) was present, pol did not reorganize into foci structures after UV irradiation, neither in cells with pan-nuclear [6Mycp21 (CDK–) left-hand panel] or focal [6Mycp21 (CDK–) right-hand panel] p21 distribution. See merged panels in supplementary material Fig. S5. (B) The percentage of cells with GFP-pol foci before and after 6 hours of UV irradiation (20 J/m2) was determined. In all cases, at least 200 transfected nuclei were counted. Values are the average and error bars are the standard deviation between equivalent samples in three independent experiments. The significance of the differences between the non-irradiated (NI) and UV samples was assessed by Student's t-test for each p21 variant (***P<0.001; **P<0.01). Further statistical analysis was performed using one-way ANOVA with Tukey-Kramer post-test (Table 1). (C) The percentage of cells with GFP-pol foci was determined at different time points after UV irradiation (20 J/m2). In all cases at least 200 transfected nuclei/sample were counted. Values are the average and error bars are the standard deviation between equivalent samples in two independent experiments.

View larger version (29K): [in this window] [in a new window]   Fig. 5. Endogenous p21 modulates pol foci assembly. (A) HCT116 p21+/+ and HCT116 p21–/– cells transfected with GFP-pol were UV irradiated (20 J/m2) when indicated. The sub-nuclear distribution of pol was determined by confocal microscopy. The percentage of cells with GFP-pol foci before and after UV irradiation was determined. In all cases, 150 transfected nuclei were counted. Values are the average and error bars are the standard deviation between equivalent samples in three independent experiments. (B) p21+/+ and p21–/– cells were subjected to UV irradiation and p21 protein levels determined using specific antibodies. Actin was used as a loading control. (C) Confocal analysis of pol organization in p21+/+ and p21–/– cells before (NT) and after UV irradiation. (D) p21–/– cells were transfected with GFP-pol and p21 or EV when indicated and UV irradiated (20 J/m2). At different times, cells were fixed and the sub-nuclear distribution of pol determined by confocal microscopy. The percentage of cells with GFP-pol foci was determined. In all cases, 150 transfected nuclei were counted. Values are the average and error bars are the standard deviation between equivalent samples in three independent experiments. (E) p21–/– cells transfected with p21 or EV were UV irradiated and p21 protein levels determined using specific antibodies. Actin was used as a loading control.

View larger version (52K): [in this window] [in a new window]   Fig. 6. The PCNA-binding domain of p21 inhibits the PCNA-pol interaction after UV irradiation. (A) U2OS cells transfected with GFP-pol and the indicated plasmids were UV irradiated. Four hours later, Triton-soluble (extractable, E) and Triton-insoluble (non-extractable, NE) fractions were collected and p21, PCNA and pol distribution in both fractions was determined using specific antibodies. (B) U2OS cells transfected with GFP-pol and the indicated plasmids were UV irradiated. Four hours later, the chromatin-bound fraction was cross-linked, sonicated and PCNA was immunoprecipitated (IP) as described in Materials and Methods. PCNA, GFP-pol , pol and p21 were detected utilizing specific antibodies. The left-hand set of panels shows PCNA IP, whereas that on the right shows an aliquot of the chromatin-bound fraction used for the PCNA IPs (INPUTS). (C) HCT116 p21+/+ and p21–/– cells (1x106) were UV irradiated and treated as in B at the indicated time points. After PCNA immunoprecipitation, endogenous pol , PCNA, pol and p21 were detected utilizing specific antibodies. Note that high exposures of the blot to film were necessary to detect p21 in the IPs shown in C.

View larger version (46K): [in this window] [in a new window]   Fig. 7. p21-PCNA interaction is detrimental for cell survival after UV irradiation. (A) U2OS cells transfected with empty vector (EV) or 6Mycp21 (CDK–) were UV irradiated (10J/m2). After fixation, immunostaining with phosphorylated H2AX (H2AX)- and p21-specific antibodies was performed. DAPI staining was used to visualize the nucleus. A complete panel showing all p21 mutants is shown in supplementary material Fig. S5. (B) U2OS cells transfected with the indicated plasmids were irradiated with 10J/m2. The number of cells with H2AX accumulation was quantified for each time point. The data shown represent the percentage of p21-positive cells with detectable accumulation of H2AX. In all cases, 100 transfected nuclei were counted. The last column in each group corresponds to the percentage of total cells with detectable accumulation of H2AX. Values are the average and error bars are the standard deviation between equivalent samples in two independent experiments. (C) U2OS cells transfected with EV or 6Mycp21 (CDK–) and GFP-pol were UV irradiated utilizing polycarbonate filters and H2AX staining used to detect the irradiated areas. (D) U2OS cells transfected with GFP-PCNA and the indicated p21 plasmids were UV irradiated (10J/m2). The cell-cycle profile of the transfected population was determined 48 hours later.

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