Werner's syndrome protein is required for correct recovery after replication arrest and DNA damage induced in S-phase of cell cycle - PubMed (original) (raw)
Werner's syndrome protein is required for correct recovery after replication arrest and DNA damage induced in S-phase of cell cycle
P Pichierri et al. Mol Biol Cell. 2001 Aug.
Free PMC article
Abstract
Werner's syndrome (WS) is a rare autosomal recessive disorder that arises as a consequence of mutations in a gene coding for a protein that is a member of RecQ family of DNA helicases, WRN. The cellular function of WRN is still unclear, but on the basis of the cellular phenotypes of WS and of RecQ yeast mutants, its possible role in controlling recombination and/or in maintenance of genomic integrity during S-phase has been envisaged. With the use of two drugs, camptothecin and hydroxyurea, which produce replication-associated DNA damage and/or inhibit replication fork progression, we find that WS cells have a slower rate of repair associated with DNA damage induced in the S-phase and a reduced induction of RAD51 foci. As a consequence, WS cells undergo apoptotic cell death more than normal cells, even if they arrest and resume DNA synthesis at an apparently normal rate. Furthermore, we report that WS cells show a higher background level of DNA strand breaks and an elevated spontaneous induction of RAD51 foci. Our findings support the hypothesis that WRN could be involved in the correct resolution of recombinational intermediates that arise from replication arrest due to either DNA damage or replication fork collapse.
Figures
Figure 1
WS cells present a higher induction of apoptosis after CPT treatment. Analysis of induction of the apoptotic cell death by pulse treatment with different doses of CPT in lymphoblast (a) and fibroblast (b) normal and WS cell lines. Normal (SNW646, AHH1, VH16, GM0842) and WS (KO375, DJG, AG00780G) cell lines were exposed to 0.1, 1, 15, and 45 μM CPT for 1 h and apoptosis evaluated 14 h later. In the figure the results from the morphological analysis by bis-benzimide staining are presented but similar results were obtained by TUNEL assay. Points represent mean ± SE from at least three experiments.
Figure 2
In WS cells CPT induces an early apoptotic response in non-S-phase cells and a later response in S-phase cells. Time course of the apoptotic induction after 1-h CPT treatment in normal (SNW646) and WS (KO375, DJG) cells (a–c); analysis of the percentage of labeled apoptotic nuclei (i.e., cells treated in the S-phase) after 1-h CPT treatment and different recovery times (d–f). Cells were pulse-labeled with BrdUrd for 30 minutes to label S-phase cells and then exposed to CPT for 1 h. Apoptotic cell death was evaluated at the indicated recovery times as described in MATERIALS AND METHODS. Because, in wild-type cells no apoptosis was detected at the earlier times after CPT (Figure 2a), the data reported in Figure 2d at 2, 4, and 6 h represent the absence of any apoptotic response rather than the absence of labeled apoptotic cells. Points represent mean from at least three experiments. Error bars were not indicated for means of clarity; SE were <10%. The horizontal reference line (a–c) represents the mean value of the spontaneous level of apoptosis in the three cell lines.
Figure 3
Example of immunocytochemical detection of BrdUrd incorporation in apoptotic nuclei. Cells were handled as described in MATERIALS AND METHODS. (A) BrdUrd-negative apoptosis (unlabeled), representing a cell that was not treated during the S-phase; note that the nucleus is only positive for the DNA. (B) BrdUrd-positive apoptosis (labeled), representing a cell treated during the S-phase.
Figure 4
Replication blockage by HU leads to the induction of apoptotic cell death in WS. Cells were exposed to 2 mM of HU for 2 h followed by different recovery periods (a). The apoptotic induction was evaluated at the indicated time by bis-benzimide staining of cells smeared onto microscopic slides as described in MATERIALS AND METHODS. Similar results were obtained by TUNEL assay; analysis of the percentage of labeled apoptotic nuclei (i.e., cells treated in the S-phase) after 2-h HU treatment and different recovery times (b). Cells were pulse-labeled with BrdUrd for 30 minutes to label S-phase cells and then exposed to HU for 2 h. Apoptotic cell death was evaluated at the indicated recovery times as described in MATERIALS AND METHODS. Points represent mean ± SE from at least three experiments.
Figure 5
WS cells arrest and resume DNA synthesis as normal cells after CPT or HU pulse treatment. Normal (SNW646) and WS (DJG) cells were exposed to 1 μM (a) or 45 μM (b) CPT for 1 h, or to 2 mM HU for 2 h (c) and recovered at the indicated times. Percentage of S-phase cells was determined labeling DNA synthesis by adding BrdUrd in the last hour before harvesting. BrdUrd incorporation was assessed as described in MATERIALS AND METHODS. Similar results were obtained also with the other normal (AHH1) and WS (KO375) cells. Points represent mean ± SE from at least three experiments.
Figure 6
Cytofluorometric bivariate analysis of the CPT-induced cell cycle perturbations. Normal, wild-type, (SNW646), and WS (KO375) cells were pulse labeled with BrdUrd for 30 min to monitor S-phase cells, washed, and then exposed to 45 μM CPT for 1 h. At the indicated recovery times cells were collected and processed for the analysis as described in MATERIALS AND METHODS. Cell cycle progression of wild-type and WS cells after CPT treatment; the horizontal and the vertical axis represent the DNA content and the BrdUrd content (i.e., cells at the S-phase during the treatment), respectively (a). Cell populations were analyzed by gating as described in b: S*, S-phase cells positive for the BrdUrd incorporation; G2*, G2 cells from the S-phase compartment positive for the BrdUrd incorporation; apo*, apoptosis (subG1 population) from S-phase cells positive for the BrdUrd incorporation; S, S-phase cells negative for the BrdUrd incorporation (i.e., G1 or G2 cells entering S-phase). (c) Percentage of cells from different stages of the cell cycle after 1-h pulse treatment with 45 μM CPT. ●, S*; □, apo*; ▴, G2*; ▿, G2. Points represent mean from at least three experiments. Error bars were not indicated for means of clarity; SE were always <10%.
Figure 7
WS cells present by Comet assay an elevated background level of DNA strand breaks and a slower repair of the CPT-induced DNA damage. Normal (SNW646, AHH1) and WS (KO375, DJG) cells were exposed to CPT for 1 h and recovered in drug-free medium for different times. At the indicated time, samples were collected and analyzed by the Comet assay as described in MATERIALS AND METHODS to obtain the time course of extinction of the CPT-induced DNA breakage (a). Similar results as reported for the SNW646 cell line were obtained with the other normal cell line (AHH1). (b) Evaluation of the spontaneous yield of DNA strand breaks in normal (SNW646, AHH1) and WS (KO375, DJG) cells, as evaluated by the tail moment analysis through Comet assay. Points represent mean ± SE from at least three experiments.
Figure 8
WS cells have an higher level of RAD51 foci but a lack of focus-forming activity after either CPT or HU treatment. Normal (SNW646, AHH1) and WS (KO375, DJG) cells were pulse exposed to CPT or HU and recovered in drug-free medium for different times. At the indicated time, samples were collected and analyzed to detect the presence of RAD51 foci as described in MATERIALS AND METHODS. (a) Representative RAD51 patterns in wild-type (A and B) and WS cells (C and D). The left column shows nuclei without RAD51 spots; the right column presents nuclei containing RAD51 focal activity. Similar focal distribution was observed either in untreated cells or after CPT and HU treatments. (b) Spontaneous level of RAD51-positive nuclei in normal and WS cells; time course of the induction of RAD51 foci after either CPT (c and d) or HU (e) treatment. Points represent mean ± SE from at least three experiments.
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