Cancer Research Annual Meeting 2010 Protein Translation and Cancer
HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
Cancer Research Clinical Cancer Research
Cancer Epidemiology Biomarkers & Prevention Molecular Cancer Therapeutics
Molecular Cancer Research Cancer Prevention Research
Cancer Prevention Journals Portal Cancer Reviews Online
Annual Meeting Education Book Meeting Abstracts Online
This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow reprints & permissions
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Rodríguez-Bravo, V.
Right arrow Articles by Agell, N.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Rodríguez-Bravo, V.
Right arrow Articles by Agell, N.
[Cancer Research 66, 8672-8679, September 1, 2006]
© 2006 American Association for Cancer Research

Cell, Tumor, and Stem Cell Biology

Chk1- and Claspin-Dependent but ATR/ATM– and Rad17-Independent DNA Replication Checkpoint Response in HeLa Cells

Verónica Rodríguez-Bravo, Sandra Guaita-Esteruelas, Roger Florensa, Oriol Bachs and Neus Agell

Departament de Biologia Cel·lular i Anatomia Patològica, Institut d'Investigacions Biomèdiques August Pi i Sunyer, Facultat de Medicina, Universitat de Barcelona, Barcelona, Spain

Requests for reprints: Neus Agell, Department Biologia Cel·lular, Facultat de Medicina, Universitat de Barcelona, C/Casanova, 143, 08036 Barcelona, Spain. Phone: 34-934035267; Fax: 34-934021907; E-mail: agell{at}medicina.ub.es.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
When DNA synthesis is inhibited, DNA replication checkpoint is activated to prevent mitosis entry without fully replicated DNA. In Xenopus, caffeine-sensitive kinases [ataxia telangiectasia mutated (ATM) and ATM-related protein (ATR)] are essential in this checkpoint response, but in mammal cells an ATR/ATM–independent checkpoint response to DNA synthesis inhibition exists. Using HeLa cells, which have a caffeine-insensitive checkpoint response, we have analyzed here which molecules known to be involved in the DNA replication checkpoint participate in the caffeine-insensitive response. When DNA synthesis was inhibited in the presence of UCN01 or after knocking down Chk1 expression [Chk1 small interfering RNA (siRNA)], HeLa cells entered into aberrant mitosis. Consequently, Chk1 is essential for both the ATR/ATM–dependent and ATR/ATM–independent checkpoint response in HeLa cells. Neither wortmannin, Ly294002, nor SB202190 abrogated the caffeine-insensitive checkpoint response, indicating that DNA-PK and p38{alpha},ß are not involved in the ATR/ATM–independent Chk1 activation upon DNA synthesis inhibition. Using siRNA to knock down Rad17 and claspin, two molecules involved in sensing stalled replication forks, we also showed that claspin but not Rad17 is essential for the ATR/ATM–independent checkpoint response. Inhibition of DNA synthesis in HeLa cells led to a decrease in cyclin B1 protein accumulation that was abrogated when UCN01 was added or when claspin was knocked down. We conclude that upon DNA synthesis inhibition, Chk1 can be activated in a claspin-dependent manner independently of ATR and ATM, leading to cyclin B1 down-regulation and providing the cells of an additional mechanism to inhibit mitosis entry. (Cancer Res 2006; 66(17): 8672-9)


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
The cell cycle must be highly regulated to ensure the complete and accurate transmission of the genome from parent to daughter cells. To this end, eukaryotic cells use checkpoints to help ensure the orderly progression and completion of critical events, such as DNA replication and chromosome segregation. As a consequence of checkpoint activation, different transitions of the cell cycle are delayed, thereby allowing time for repair processes or completion of critical cell cycle events (1). The fundamental importance of these cell cycle checkpoints is underscored by the genetic instability that results from the loss of checkpoint functions and the role such defects play in the evolution of normal cells to cancer cells (2–6).

All checkpoints function via the action of (a) sensor molecules that monitor or sense different cell cycle events or the presence of DNA damage; (b) mediators, which transduce signals; and (c) effectors that are responsible for cell cycle arrest and any necessary repairs. Some molecules seem to function as both sensors and mediators. The most closely studied checkpoints are those induced by DNA damage (7, 8). Key upstream proteins involved in DNA damage response include ataxia telangiectasia mutated (ATM) and ATM-related protein (ATR), two serine/threonine kinase members of a phosphoinositide 3-kinase-like family (9). Activation of these kinases results in the phosphorylation of a diverse array of downstream targets that participate in numerous cellular events, including DNA damage recognition and processing, cell cycle arrest at different points depending on when such DNA damage is detected, and apoptosis. Chk1 and Chk2, two mediator kinases activated by ATM and ATR, are involved in cell cycle arrest response (10). Although not as well studied, the DNA replication checkpoint is also very important in preventing genetic integrity. In the presence of stalled replication forks, cells activate a checkpoint response to stabilize the arrested forks and delay initiation of late origins (the intra-S checkpoint pathway) and also inhibit mitosis entry (the S-M checkpoint pathway). Although the exact mechanism of this checkpoint is not clearly understood, it features elements common to that activated by DNA damage detection during the S phase. This mechanism has typically been analyzed by treating cells with hydroxyurea, thereby inhibiting ribonucleotide reductase and therefore DNA synthesis. DNA damage induced by UV light or methyl methanesulfonate treatment blocks DNA replication fork progression, consequently activating the DNA replication checkpoint (11, 12). How partially replicated DNA is detected remains unknown, but several studies suggest that the signal arises from the replication machinery itself (13–15). Three different molecules seem to act as sensors, independently binding to stalled replication forks. First, ATR complexed to ATRIP recognizes long ssDNA segments covered by RPA that appear when DNA synthesis is arrested (16, 17), although RPA-independent binding of ATR to DNA has also been reported (18). Second, Rad17 interacts with regions of DNA polymerase {alpha}–primed DNA, subsequently acting as a clamp loader of the Rad9-Rad1-Hus1 complex (19, 20). Third, claspin, which binds independently of RPA, ATR, and Rad17 to chromatin (21), has recently been shown to bind to branched structures also present in the DNA replication forks (22). Once loaded to DNA, Rad17 and claspin collaborate to fully activate ATR. The latter will then phosphorylate different substrates, including Rad17 itself (20, 23), Chk1 (24, 25), and SMC-1 (which is required for sister chromatid cohesion and recombination; ref. 26). Claspin also binds to Chk1 and is essential for ATR phosphorylation of Chk1, therefore acting both as a sensor and mediator molecule for the DNA replication checkpoint (27). There is increasing evidence that this entire process occurs not only in response to stalled replication forks, but also during the normal S phase, albeit with a lower intensity, to inhibit both mitosis entry and late origin firing while DNA synthesis remains ongoing (28, 29). Chk1 plays a central role in the checkpoint response (30), phosphorylating Cdc25A and Cdc25C (31). On one hand, phosphorylation of Cdc25A increases its degradation and, consequently, inactivation of cdk2. This phosphorylation-induced degradation of Cdc25A also occurs in response to DNA damage during the S phase (8, 32). In this case, Cdk2 inhibition leads not only to ongoing DNA synthesis arrest, but also to inhibition of late origin replication firing. On the other hand, Cdc25C phosphorylation has been shown to increase both its turnover and cytoplasmic retention (33–35). Consequently, activation of the Cdc2/cyclin B1 complex is prevented and mitosis entry is inhibited. Although in many organisms, such as yeast and Xenopus, caffeine-sensitive kinases (ATM or ATR) are essential to the DNA replication checkpoint, this is not the case with all mammalian cell lines. The presence of an ATR- and ATM-independent checkpoint response to DNA replication has been shown in mammalian cells using either ATR/ATM inhibitors or a genetic approach (36, 37). One of the consequences of this response was an inhibition of cyclin B1 accumulation and consequently a decrease in Cdc2/cyclin B1 activity and, therefore, a blockade of mitosis entry (36). Interestingly, although a caffeine-insensitive response exists in all nontransformed cell lines analyzed and in some transformed cell lines such as HeLa, it was found altered in most colorectal cancer–derived cell lines, suggesting that its disruption may be relevant to carcinogenesis. This prompted us to examine which molecules could be involved in mediating the novel ATR/ATM–independent checkpoint response to the DNA synthesis inhibition. Because of the central role of Chk1 in the DNA replication checkpoint, we first analyzed its possible participation in the caffeine-insensitive response. Furthermore, because Rad17 and claspin independently bind to chromatin following DNA synthesis inhibition, we sought to determine whether either could also independently signal to activate an ATR-independent checkpoint response.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Drugs. Hydroxyurea and caffeine (Sigma, St. Louis, MO) stocks were prepared in water at 300 and 100 mmol/L, respectively. UCN01 (National Cancer Institute, Bethesda, MA), Ly294002 (Calbiochem, Darmstadt, Germany), wortmannin (Calbiochem), SB202190 (Sigma), cisplatin (Sigma), and anysomicine (Sigma) were all dissolved in DMSO at concentrations of 300 µmol/L, 20 mmol/L, 50 mmol/L, 5 mg/mL, 15 mmol/L, and 2 mg/mL respectively. Working concentrations were 1.5 mmol/L for hydroxyurea, 5 mmol/L for caffeine, 300 nmol/L for UCN-01, 25 µmol/L for Ly294002, 30 or 200 µmol/L for wortmannin to inhibit DNA-PK or all phosphatidylinositol 3-kinases (PI3K), respectively, and 20 µmol/L for SB 202190. Cisplatin working concentration was 15 µmol/L and for anysomicine was 1 µg/mL. All stocks were stored at –20°C.

Cell culture. HeLa cells were obtained from the European Collection of Cell Cultures. HCT116 cells were a gift from Dr. Capellà (Institut Català d'Oncologia, Barcelona, Spain). HeLa cells were grown in DMEM supplemented with 10% FCS. HCT116 cells were grown in DMEM:HAM F12 (1:1) supplemented with 10% FCS.

HeLa cells growing in 100-mm dishes were synchronized by double thymidine block. Cells at 40% confluency were treated with 2.5 mmol/L thymidine in DMEM supplemented with 10% FCS for 24 hours. Cells were then washed twice with PBS and incubated for 12 hours more in DMEM 10% FCS in the absence of thymidine. After this recovery, step cells were incubated a second time with 2.5 mmol/L thymidine in DMEM 10% FCS for 24 hours to arrest cells at the G1-S boundary of the cell cycle. Cells synchronized in G1-S were washed twice with PBS and then released back into the cell cycle (0 hour point).

Gel electrophoresis and immunoblotting. Cells were lysed in a buffer containing 2% SDS, 67 mmol/L Tris-HCl (pH 6.8), and sonicated twice for 20 seconds. Protein content was measured by the Lowry procedure, using bovine serum albumin (BSA) as a standard. These cell extracts were electrophoresed in SDS-polyacrylamide gels, essentially as previously described (38). Following electrophoresis, the proteins were transferred to a polyvinylidene difluoride membrane (Immobilon-P, Millipore) for 2 hours at 60 V. The sheets were preincubated in TBS [20 mmol/L Tris-HCl (pH 7.5), 150 mmol/L NaCl], 0.05% Tween 20, and 5% defatted powdered milk for 1 hour at room temperature and then incubated in TBS, 0.05% Tween 20, and 1% defatted milk powder containing the appropriate antibodies. Incubation with Rad17 (Santa Cruz Biotechnology, Santa Cruz, CA; 1:500), claspin (Bethyl Laboratories; 1:5,000 dilution), actin (MP Biomedicals; 1:10,000 dilution), and Chk1 (Santa Cruz Biotechnology; 1:300), was done for 1 hour at room temperature. Incubation with phospho-Chk1 Ser345 (Cell Signalling Technology; 1:1,000), MAPKAPK2 (Cell Signaling Technology; 1:1,000), phospho-MAPKAPK2 Thr334 (Cell Signaling Technology; 1:1,000), phospho-MAPKAPK2 Thr222 (Cell Signaling Technology; 1:1,000), and cyclin B1 (Santa Cruz Biotechnology; 1:100) antibodies was conducted overnight at 4°C. After washing in TBS, 0.05% Tween 20 (thrice, 10 minutes each), the sheets were incubated with the appropriate peroxidase-coupled secondary antibody (Bio-Rad; 1:2,000 dilution) for 1 hour at room temperature. Following incubation, the sheets were washed thrice in TBS, 0.05% Tween 20, and then twice in TBS. The reaction was observed using enhanced chemiluminescence (ECL), ECL Advance (Amersham), and EZ-ECL (Biological Industries) as reagents.

Immunocytochemistry and laser scanning cytometry analysis. Cells were grown on 12-mm coverslips and following treatment were washed twice with PBS, fixed with ethanol/acetic acid (95:5) for 5 minutes at room temperature, and then blocked in 8% BSA for 1 hour at room temperature. Cells were subsequently incubated with the primary antibody against phospho-histone H3 (Ser10; Upstate Biotechnology; 1:200 dilution) for 1 hour at 37°C. Mitotic cells were detected with a secondary Alexa 488–conjugated antibody (1:500 dilution; The Jackson Laboratory). The total number of cells was quantified by DNA staining with propidium iodide (1 µg/mL) following RNase A (20 µg/mL) treatment. At least 10,000 events were recorded by the cytometer for each sample. An argon laser set at 5 mW was used to excite the fluorochromes, and the filters used were 530/30 nm for Alexa 488 and 625/28 nm for propidium iodide.

Flow cytometry. Synchronized cells were trypsinized, washed with PBS, and centrifuged for 5 minutes at 2,000 rpm. Pelleted cells were resuspended in 500 µL PBS, fixed with 4.5 mL cold (–20°C) 70% ethanol, and stored at 4°C. Cells were then washed with PBS, centrifuged, and incubated with 20 µg/mL propidium iodide (Sigma) dissolved in PBS with 0.2 mg/mL RNase A (Sigma) and 0.1%(v/v) Triton X-100 (Sigma) for 30 minutes at room temperature. DNA content was measured by flow cytometry (FACScan Analyzer; Becton Dickinson) and analyzed using the Sync Wizard Model, ModFit LT software (Becton Dickinson). Aggregated cells revealed by forward scattering were filtered out of the data set before analysis.

Small interfering RNA. HeLa cells were grown for 1 day on glass coverslips and transfected with the small interfering RNA (siRNA) SMARTpools of Chk1, Rad17 (Dharmacon), claspin (Dharmacon), Chk1 duplex siRNA (5'-GCGUGCCGUAGACUGUCCAdTdT-3'; Dharmacon), or claspin duplex siRNA (5'-GACUCAGCUCUAAGCAAGGdTdT-3'; Dharmacon) using Lipofectamine 2000 (Invitrogen) following the instructions of the manufacturer. Control experiments were done, transfecting the cells with a green fluorescent protein (GFP) duplex siRNA (NNGGCUACGUCCAGGAGCGCA sequence; Dharmacon). Forty-eight hours after transfection, Western blot analysis indicated that levels of Chk1, Rad17, or claspin were drastically reduced in those cells transfected with siRNA of Rad17 or claspin, respectively, but not in control siRNA (data not shown). Cell lysates were obtained and coverslips were fixed as previously described.

Colony formation assay. HeLa cells were trypsinized, counted, and seeded in 60-mm dishes (1,000 per dish). The next day, cells were pretreated with 25 µmol/L Ly294002 or 30 µmol/L wortmannin for 1 hour. Cisplatin was then added at a final concentration of 15 µmol/L for 1 hour. Ly294002 or wortmannin were maintained in the culture medium for the next 16 hours and, finally, medium was changed and the cells kept growing in fresh medium without any drug during 10 days. After that time, colonies were fixed, stained with crystal violet solution, and counted.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Involvement of Chk1 in the ATR/ATM–independent checkpoint response to DNA synthesis inhibition in HeLa cells. We previously showed that nontransformed cell lines and some tumor cell lines, such as HeLa cells, have an ATR/ATM–independent checkpoint response to DNA synthesis inhibition, which is not functional in most colorectal cancer cells lines analyzed, like, for instance, in HCT116 cells. Using HeLa cells, we sought to determine first whether Chk1, a key element in the ATR/ATM–dependent response of the DNA replication checkpoint, was also participating in the ATR/ATM–independent response. To this end, asynchronic populations of HeLa and HCT116 cells were treated with hydroxyurea for 25 hours to inhibit DNA synthesis, and during the last 10 hours of treatment caffeine (ATM and ATR inhibitor) or UCN01 (Chk1 inhibitor) or both were also added to the medium. The percentage of cells entering mitosis was determined as the percentage of phospho-H3–positive cells by immunostaining. Confirming our previous results, whereas caffeine abrogated the checkpoint response induced by hydroxyurea in HCT116 cells, HeLa cells still inhibited mitosis entry upon treatment with hydroxyurea plus caffeine (Fig. 1A ). Although a controversy exist relating the ability of caffeine to inhibit phosphorylation of ATM and ATR targets (39), caffeine addition reduced, under our experimental conditions, Chk1 phosphorylation at Ser345 and at Ser317, indicating that caffeine was, in fact, inhibiting ATR (Fig. 1B). Therefore, in HeLa, but not in HCT116 cells, a checkpoint response was observed even in the presence of very low levels of Chk1 phosphorylated at Ser345 or Ser317. Furthermore, addition of 200 µmol/L wortmannin, a concentration that is inhibitory of ATR and ATM (40), did not abrogate the checkpoint response in HeLa cells (data not shown). Interestingly, UCN01 abrogated the mitosis entry inhibition induced by hydroxyurea in both HeLa and HCT116, suggesting that Chk1 activity was essential for the checkpoint response in both cell lines (Fig. 1A). Thus, cells treated with hydroxyurea and UCN01 entered mitosis without finishing DNA replication. Consistent with that, all mitosis observed (phospho-H3–positive cells with condensed chromatin) were abnormal (data not shown). To further confirm the indispensable role of Chk1 in this checkpoint response, Chk1 knockdown was conducted using the siRNA technique. As shown in Fig. 2A , following a treatment with a pool of four siRNA against Chk1, Chk1 protein levels decreased drastically and correlated with an abrogation of the checkpoint response to hydroxyurea. To be sure of the specificity of the siRNA treatment, the same experiment was done using a single RNA duplex to knockdown Chk1. Levels of Chk1 did not decrease so drastically, but the checkpoint was still abrogated, especially when cells were treated with caffeine. In the absence of caffeine, checkpoint abrogation was not very strong using this RNA duplex. Maybe the presence of low levels of Chk1 when using the single RNA duplex still allowed some arrest. Nevertheless, our data clearly show that an ATR- and ATM-independent mechanism of Chk1 activation, which most probably does not require phosphorylation of Chk1 at Ser345 or Ser317, should exist in these cells.


Figure 1
View larger version (21K):
[in this window]
[in a new window]
  		Figure 1. Checkpoint response to hydroxyurea addition is abrogated by UCN01 but not by caffeine in HeLa cells. A, asynchronously growing HCT116 and HeLa cells were treated, when indicated, with 1.5 mmol/L hydroxyurea (HU) for 25 hours. Also, where indicated, 5 mmol/L caffeine (HU+Caff), 300 nmol/L UCN01 (HU+UCN01), or both (HU+Caff+UCN01) were added during the last 10 hours of hydroxyurea treatment. Cells not treated with hydroxyurea were treated only for 10 hours with either caffeine or UCN01. In control cells, no drug was added. After treatments, cells were fixed and percentage of phospho-H3–positive cells (%P-H3) analyzed as indicated in Materials and Methods. B, asynchronously growing HCT116 and HeLa cells were treated with 1.5 mmol/L hydroxyurea for 25 hours. Where indicated, caffeine was added at different concentrations during the last 10 hours of hydroxyurea treatment. After treatment, cells were harvested and the same amount of total protein lysate was electrophoresed, and Chk1 phosphorylated at Ser345 (Chk1S345P) or at Ser317 (Chk1S317P) was analyzed by Western blotting using anti-phospho-Ser345-Chk1– or anti-phospho-Ser317-Chk1–specific antibodies. Western blots against total Chk1 or actin were used as loading control. Untreated cells (–) and cells treated only with caffeine for 10 hours were also analyzed. When not indicated, caffeine concentration was 5 mmol/L.

 

Figure 2
View larger version (15K):
[in this window]
[in a new window]
  		Figure 2. Chk1 knockdown abrogates hydroxyurea-induced checkpoint response in HeLa cells. HeLa cells were transfected as indicated in Materials and Methods with siRNA Smart Pool for Chk1 [Chk1i (pool); A] or with a siRNA duplex for Chk1 [Chk1i (dplx); B], or an unrelated siRNA as control (GFPi; both A and B). Where indicated, 48 hours after transfection, cells were treated with hydroxyurea for 25 hours, and caffeine (HU Caff) or nothing (HU) was added for the last 10 hours. In those cells not treated with hydroxyurea, caffeine (Caff) or nothing (–) were added for 10 hours at the same time as the other cells (63 hours after transfection). After treatments, cells were fixed and the percentage of phospho-H3–positive cells was analyzed as indicated in Materials and Methods; cells were lysed and the amount of total Chk1 was analyzed by Western blotting. Actin was used as loading control. One representative experiment out of three (A and B).

 
DNA-PK, as ATR and ATM, is a member of the PI3K family of protein kinases, and it participates in DNA damage response, but it is not inhibited by caffeine. Wortmannin and Ly294002, both inhibitors of the PI3K family, were used to test whether DNA-PK was participating in the ATR/ATM–independent checkpoint response to hydroxyurea treatment. Although IC50 of wortmannin is 16 nmol/L for DNA-PK, 150 nmol/L for ATM, and 1.8 µmol/L for ATR, in intact cells the doses used to inhibit DNA-PK range between 10 and 30 µmol/L (40). Similarly, although the described IC50 of Ly294002 for DNA-PK is lower than 1 µmol/L (41), the dose used to ensure inhibition of DNA-PK in HeLa cells is 25 µmol/L (42). Consequently, HeLa cells were treated for 25 hours with hydroxyurea, being 25 µmol/L Ly294002 or 30 µmol/L wortmannin added for the last 10 hours, alone or together with caffeine. The functionality of the checkpoint response in the presence of those inhibitors was analyzed as before quantifying the percentage of phospho-H3–positive cells by immunocytochemistry. As shown in Fig. 3A , none of the drugs alone or in combination with caffeine abrogated the mitosis entry arrest induced by hydroxyurea treatment. In contrast, both 30 µmol/L wortmannin or 25 µmol/L Ly294002 sensitized HeLa cells to cisplatin treatment (Fig. 3B), indicating that they were in fact inhibiting DNA-PK. Using SB202190, an inhibitor of p38{alpha} and p38ß, we also tested the involvement of this MAPK, which has also been shown to participate in the checkpoint response induced by UV irradiation (Fig. 3A). This drug was also not able to abrogate the checkpoint response either alone or in combination with caffeine. As a control to test that SB202190 was in fact inhibiting p38 activity in our cells, we showed that its addition inhibited the hydroxyurea- and anysomicine-induced phosphorylation of MAPKAPK2, a p38 substrate (Fig. 3C). In conclusion, neither DNA-PK nor p38{alpha} and p38ß were essential in the ATR/ATM–independent checkpoint response to hydroxyurea treatment.


Figure 3
View larger version (23K):
[in this window]
[in a new window]
  		Figure 3. The caffeine-insensitive checkpoint response to hydroxyurea of HeLa is not abrogated by PI3K or p38 {alpha} and ß inhibitors. A, HeLa cells were treated with 1.5 mmol/L hydroxyurea for 25 hours. During the last 10 hours, the indicated drug combinations were added. For cells not treated with hydroxyurea, the indicated drugs were added for 10 hours. Caff, caffeine 5 mmol/L; SB, SB202190 20 µmol/L; Wo, wortmannin 30 µmol/L; Ly, Ly294002 25 µmol/L; C, untreated cells. After treatments, cells were fixed and the percentage of phospho-H3–positive cells was analyzed as indicated in Materials and Methods. B, control of wortmannin and Ly294002 functionality. Cell survival of HeLa cells after 15 µmol/L cisplatin treatment alone (Cis) or in the presence of 30 µmol/L wortmannin (Cis+Wo) or 25 µmol/L Ly294002 (Cis+Ly); without any treatment (Cont); or treatment with wortmannin or Ly294002 alone was analyzed by colony formation assay as shown in Materials and Methods. C, control of SB202190 functionality. HeLa cells were treated with 1 µg/µL anysomicine (Anys) ± 20 µmol/L SB202190 for 10 minutes (left) or with 1.5 mmol/L hydroxyurea ± 20 µmol/L SB202190 for 15 hours (right). Then, the cells were lysed, and total MAPKAPK2 (MK2) and MAPKAPK2 phosphorylated at Thr344 (MK2T344) and at Thr222 (MK2T222) were analyzed by Western blotting as shown in Materials and Methods.

 
The ATR/ATM–independent checkpoint response to DNA synthesis inhibition is RAD17-independent but claspin-dependent in HeLa cells. As ATR-ATRIP, Rad17, and claspin all bind independently to chromatin upon DNA replication arrest, we wanted to determine whether Rad17 or claspin could signal independently of ATR to activate a DNA replication checkpoint response in HeLa cells. To this end, protein knockdown was conducted using the siRNA technique. As shown in Fig. 4A , following Rad17 siRNA treatment, protein levels decreased drastically whereas the percentage of cells entering mitosis upon hydroxyurea treatment was not affected, either in the absence or presence of caffeine. This indicated that the ATR-independent response was also Rad17-independent. Interestingly, inhibition of claspin expression by claspin-specific siRNA treatment did not abrogate the cell cycle arrest induced by hydroxyurea in the absence of caffeine, indicating that claspin was dispensable when ATR or ATM were active. However, when cells were simultaneously treated with hydroxyurea plus caffeine, the absence of claspin completely abrogated the response and the percentage of cells entering mitosis increased drastically (Fig. 4B). The same results were obtained using a single siRNA duplex for claspin, nonrelated with the ones found in the used pool (data not shown). This showed that the caffeine-insensitive response to DNA replication checkpoint activation was claspin dependent.


Figure 4
View larger version (16K):
[in this window]
[in a new window]
  		Figure 4. Effect of Rad17 and claspin knockdown on hydroxyurea-induced checkpoint response in HeLa cells. HeLa cells were treated as indicated in Materials and Methods with siRNA to knockdown Rad17, Rad17i (A) or claspin, claspin i, (B) expression. In both cases, a control with a nonrelated siRNA was also done (GFPi). Where indicated, 48 hours after transfection, cells were treated with hydroxyurea for 25 hours, and caffeine (HU+Caff) or nothing (HU) was added for the last 10 hours. In cells not treated with hydroxyurea, caffeine (Caff) or nothing (–) was added for 10 hours at the same time as the other cells (62 hours after transfection). After treatments, cells were fixed and the percentage of phospho-H3–positive cells was analyzed as indicated in Materials and Methods (top) or lysed and the amount of total Rad17 or claspin was analyzed by Western blotting (bottom). Actin was used as loading control. One representative experiment out of three (A and B).

 
Claspin and Chk1 down-regulate cyclin B1 levels in HeLa cells. We have previously shown that when cells with a functional ATR/ATM–independent response to DNA synthesis inhibition were treated with hydroxyurea plus caffeine, Cdc2/cyclin B1 activity remained low and this correlated with low cyclin B1 levels. In contrast, in cells whose caffeine-insensitive checkpoint response was not functional, cyclin B1 levels were high upon DNA synthesis inhibition (36). We hypothesized that blockade of DNA replication inhibited cyclin B1 accumulation, and, consequently, Cdc2/cyclin B1 activity and mitosis entry in an ATR- and ATM-independent manner.

We show now that this is also the case in HeLa cells. HeLa cells were synchronized by double thymidine block at G1-S transition and, 2 hours upon release, hydroxyurea was added to block DNA synthesis (Fig. 5A ). After 6 hours, control cells (which were in G2 or M phase) or hydroxyurea-treated cells were harvested and levels of cyclin B1 were analyzed. As shown in Fig. 5A and B, hydroxyurea-treated cells did not duplicate DNA and did not accumulate cyclin B1. Caffeine addition did not abrogate the hydroxyurea effect on cyclin B1 accumulation, whereas upon UCN01 treatment, cyclin B1 increased as in control cells. Consequently, Chk1, but not ATR, was essential to inhibit cyclin B1 accumulation upon hydroxyurea treatment in HeLa cells. Furthermore, as with asynchronic cells, UCN01 allowed mitosis entry (phosho-H3–positive cells), whereas caffeine treatment, which did not induced cyclin B1 accumulation, did not (Fig. 5C). As expected, the phospho-H3–positive cells observed in hydroxyurea plus UCN01–treated cells corresponded to abnormal mitosis, because those cells did not completely replicate the DNA (Fig. 5A and C). We showed above that claspin is also an essential component of the checkpoint response in HeLa cells; therefore, we subsequently analyzed whether it was involved in the down-regulation of cyclin B1. As shown in Fig. 6 , a strong increase in cyclin B1 expression was observed upon claspin depletion using siRNA. Furthermore, this inhibitory role of claspin on cyclin B1 expression was observed even in the presence of caffeine, indicating that it was ATR and ATM independent. Higher levels of cyclin B1 were also observed upon hydroxyurea or hydroxyurea plus caffeine treatment in cells depleted for claspin compared with nondepleted ones (data not shown).


Figure 5
View larger version (29K):
[in this window]
[in a new window]
  		Figure 5. Inhibition of cyclin B1 accumulation induced by hydroxyurea treatment in HeLa is abrogated by UCN01 but not by caffeine treatment. HeLa cells were synchronized by a double thymidine block as indicated in Materials and Methods. Two hours after thymidine block release, cells were treated with the indicated drug combination for 6 hours more. After the different treatments, the following analyses were done. A, fluorescence-activated cell sorting showing DNA content profile after the different treatment. B, Western blotting to determine the total amount of cyclin B1. The same amount of protein was loaded in all lines and actin was used as loading control. C, propidium iodide (PI) staining and immunocytochemistry to detect phospho-H3–positive cells. Representative fluorescent microscope fields. D, two representative mitoses stained with phospho-H3 antibody: 2h+6h, normal mitosis of a HeLa cells nontreated with any drug; 2h+6h HU+UCN01, abnormal mitosis of a HeLa cell that entered mitosis with not fully replicated DNA.

 

Figure 6
View larger version (7K):
[in this window]
[in a new window]
  		Figure 6. Claspin knockdown induced an increase of cyclin B1 in HeLa cells. HeLa cells were transfected for 72 hours with siRNA to knockdown claspin (Claspin i) expression or with a nonrelated siRNA as control (GFPi). For the last 10 hours, cells were treated (Caff) or not (–) with 5 mmol/L caffeine. The cells were lysed, and the amount of cyclin B1 and claspin was analyzed by Western blotting. Then, the same amount of protein was loaded in all lines and actin was used as loading control.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
When DNA synthesis is arrested, a checkpoint response is activated, thereby delaying the cell cycle, stabilizing arrested replication forks, and activating repair of any possible DNA damage, all of which are essential to maintain genome integrity. In Xenopus and yeast, this response has been shown to be completely dependent on either ATR and ATM kinases, or their yeast homologues (1). In contrast, in mammalian nontransformed cells, our group and others have shown that a corresponding ATR/ATM–independent pathway exists (36, 37). The existence of this pathway may help cells to survive the inhibition of the ATR/ATM pathway. We have also previously shown that this pathway is not functional in most of the colorectal cancer cells analyzed (36), but functional in other tumor cell lines like some tumor pancreas-derived cells and in HeLa cells. Using HeLa cells, we have analyzed the involvement of other checkpoint proteins in the ATR/ATM–independent response. We first corroborated that upon DNA synthesis inhibition by hydroxyurea treatment, caffeine addition abrogated the checkpoint response in HCT116 cell but not in HeLa cells. The fact that upon caffeine treatment both cell lines had very low levels of Chk1 phosphorylated at Ser345 and Ser317 but only HeLa cell inhibited mitosis entry indicated that HeLa cells had a DNA replication checkpoint response that was independent of ATR and ATM and of Chk1 phosphorylation at Ser345 and Ser317. However, by using UCN01 as Chk1 inhibitor or siRNA to knock down Chk1 expression, we showed that this kinase is an essential element in the checkpoint response to hydroxyurea and participates in both the ATR-dependent and ATR-independent pathways. In both UCN01 and siRNA Chk1 treatment, the checkpoint was abrogated specially in the presence of caffeine. Abrogation of the checkpoint induced mitosis entry with not fully replicated DNA and, consequently, all mitoses were aberrant. This results are consistent with the fact that Chk1 inhibition potentiates in HeLa cells the apoptosis induced by 5-fluorouracil, an antimetabolite that inhibits DNA synthesis and is widely used in the treatment of diverse cancers (43). Although the manner in which cells monitor the presence of stalled replication forks is not completely clear, the following elements seem essential: (a) RPA, which binds to ssDNA and then recruits ATRIP and ATR (16, 17); (b) Rad17, which recognizes RNA primers and then loads the Rad9-Rad1-Hus1 complex to DNA (20); and (c) claspin, which recognizes branched DNA structures (19, 22). All of these molecules are thus important to fully activate the ATR-mediated checkpoint response leading to the phosphorylation of ATR substrates, and finally to cell cycle arrest and stabilization of replication forks (7). Here, we have analyzed whether some of the proteins involved in monitoring stalled replication forks may also mediate the caffeine-insensitive response observed in HeLa cells. In the absence of Rad17, cells could still inhibit mitosis in response to hydroxyurea, either in the presence or absence of caffeine, indicating that Rad17 was dispensable for caffeine-sensitive and caffeine-insensitive responses. Activation of ATM is likely the explanation why Rad17 is not essential for the checkpoint response in the absence of caffeine. Because hydroxyurea treatment was extended, DNA damage could be occurring and, consequently, both kinases ATR and ATM could be activated. When caffeine was added, however, both pathways were inhibited but cells were still arrested. In the absence of claspin, however, although cells could still inhibit mitosis when DNA synthesis was inhibited and ATR and ATM active, they were unable to do so when those same kinases were inhibited with caffeine. This clearly indicates that claspin is essential for caffeine-insensitive checkpoint responses. Thus, our results indicate that claspin, but not Rad17, is involved in the caffeine-insensitive checkpoint response. We therefore hypothesize that when DNA synthesis is inhibited, claspin bound to the stalled replication forks interacts with Chk1 and allows, as already described, its phosphorylation at Ser345 and Ser317 by ATR, but also its activation by maybe another kinase (Fig. 7 ). Our results exclude the participation of DNA-PK and p38, two kinases known to be activated upon DNA damage, in the caffeine-insensitive checkpoint response to DNA synthesis inhibition; consequently, these two kinases are not likely to participate in ATR/ATM–independent Chk1 activation. Chk1 activated by ATR leads to Cdc25 inhibition as already described, but ATR/ATM–independent, claspin-dependent activation of Chk1 would lead to an inhibition of cyclin B1 accumulation and consequently to an inhibition of Cdk1/cyclin B1 and mitosis entry. Consistent with this hypothesis, we showed first that the lack of cyclin B1 accumulation in S-phase HeLa cells treated with hydroxyurea is abrogated by UCN01 treatment but not by caffeine. Furthermore, we also showed in the present study a link between claspin levels and cyclin B1 expression, because knocking down claspin induced increases in cyclin B1 independently of ATR/ATM activity. Association with different protein complexes could explain the differential functionality of Chk1 activated by ATR or by a distinct unknown kinase. We therefore propose that the binding of claspin to replication forks leads, through a Chk1-dependent but ATR- and ATM-independent pathway, to the inhibition of cyclin B1 expression, and that this inhibition is released as replication forks disappear at the end of the S phase, or is reinforced when DNA synthesis is arrested.


Figure 7
View larger version (21K):
[in this window]
[in a new window]
  		Figure 7. Model for DNA replication checkpoint response in HeLa cells. The model shows the classic ATR-, Rad17-, and claspin-dependent activation of Chk1 and the ATR- and Rad17-independent but claspin-dependent Chk1 activation described in the present work. ATR-dependent activation leads to Chk1 phosphorylation at Ser345/317 and to an inhibition of Cdc25 as already described. We propose that the ATR-independent activation of Chk1 (Chk1*) does not correlate with its phosphorylation at Ser345/317 and leads to a decrease in cyclin B1 accumulation, providing the cell with an additional mechanism to inhibit mitosis entry. This response is not functional in HCT116 cells. CycB, cyclin B1; 9-1-1, Rad9-Rad1-Hus1 complex.

 
ATR-, Rad17-independent but Chk1-dependent checkpoint response to DNA synthesis inhibition provides the cell with an additional mechanism to inhibit mitosis entry, ensuring genomic stability. In fact, this response is not functional in most colorectal cancer cells like HCT116 cells (36). Chk1 inhibitors have been proposed as good anticancer drugs, especially when used in combination with DNA-damaging agents (44). The fact that this kinase is essential for both ATR-dependent and ATR-independent checkpoint response to DNA synthesis inhibition reinforces the idea of using Chk1 inhibitors in cancer therapy but also in combination with drugs that inhibit DNA synthesis.


   Acknowledgments
 
Grant support: Grants SAF2001-2901, SAF2004-02159, and GEN2003-20243-C08-01; and a predoctoral fellowship from CIRIT (Commissìó Interdepartamental de Recerca i Innovació Tecnològica) (Catalan government; V. Rodríguez).

The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Received 12/13/05. Revised 5/31/06. Accepted 6/29/06.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

  1. Nyberg KA, Michelson RJ, Putnam CW, Weinert TA. Toward maintaining the genome: DNA damage and replication checkpoints. Annu Rev Genet 2002;36:617–56.[CrossRef][Medline]
  2. Hartwell L. Defects in a cell cycle checkpoint may be responsible for the genomic instability of cancer cells. Cell 1992;71:543–6.[CrossRef][Medline]
  3. Kastan MB, Bartek J. Cell-cycle checkpoints and cancer. Nature 2004;432:316–23.[CrossRef][Medline]
  4. Hartwell LH, Kastan MB. Cell cycle control and cancer. Science 1994;266:1821–8.[Abstract/Free Full Text]
  5. Elledge SJ. Cell cycle checkpoints: preventing an identity crisis. Science 1996;274:1664–72.[Abstract/Free Full Text]
  6. Hartwell L. Defects in a cell cycle checkpoint may be responsible for the genomic instability of cancer cells. Cell 1999;71:543–6.
  7. Sancar A, Lindsey-Boltz LA, Unsal-Kacmaz K, Linn S. Molecular mechanisms of mammalian DNA repair and the DNA damage checkpoints. Annu Rev Biochem 2004;73:39–85.[CrossRef][Medline]
  8. Bartek J, Lukas C, Lukas J. Checking on DNA damage in S phase. Nat Rev Mol Cell Biol 2004;5:792–804.[CrossRef][Medline]
  9. Shiloh Y. ATM and related protein kinases: safeguarding genome integrity. Nat Rev Cancer 2003;3:155–68.[CrossRef][Medline]
  10. Bartek J, Lukas J. Chk1 and Chk2 kinases in checkpoint control and cancer. Cancer Cell 2003;3:421–9.[CrossRef][Medline]
  11. Stokes MP, Van Hatten R, Lindsay HD, Michael WM. DNA replication is required for the checkpoint response to damaged DNA in Xenopus egg extracts. J Cell Biol 2002;158:863–72.[Abstract/Free Full Text]
  12. Lupardus PJ, Byun T, Yee MC, Hekmat-Nejad M, Cimprich KA. A requirement for replication in activation of the ATR-dependent DNA damage checkpoint. Genes Dev 2002;16:2327–32.[Abstract/Free Full Text]
  13. Michael WM, Ott R, Fanning E, Newport J. Activation of the DNA replication checkpoint through RNA synthesis by primase. Science 2000;289:2133–7.[Abstract/Free Full Text]
  14. You Z, Kong L, Newport J. The role of single-stranded DNA and polymerase {alpha} in establishing the ATR, Hus1 DNA replication checkpoint. J Biol Chem 2002;277:27088–93.[Abstract/Free Full Text]
  15. D'Urso G, Grallert B, Nurse P. DNA polymerase {alpha}, a component of the replication initiation complex, is essential for the checkpoint coupling S phase to mitosis in fission yeast. J Cell Sci 1995;108:3109–18.[Abstract]
  16. Cortez D, Guntuku S, Qin J, Elledge SJ. ATR and ATRIP: partners in checkpoint signaling. Science 2001;294:1713–6.[Abstract/Free Full Text]
  17. Zou L, Elledge SJ. Sensing DNA damage through ATRIP recognition of RPA-ssDNA complexes. Science 2003;300:1542–8.[Abstract/Free Full Text]
  18. Kim SM, Kumagai A, Lee J, Dunphy WG. Phosphorylation of Chk1 by ATM- and Rad3-related (ATR) in Xenopus egg extracts requires binding of ATRIP to ATR but not the stable DNA-binding or coiled-coil domains of ATRIP. J Biol Chem 2005;280:38355–64.[Abstract/Free Full Text]
  19. Lindsey-Boltz LA, Bermudez VP, Hurwitz J, Sancar A. Purification and characterization of human DNA damage checkpoint Rad complexes. Proc Natl Acad Sci U S A 2001;98:11236–41.[Abstract/Free Full Text]
  20. Zou L, Cortez D, Elledge SJ. Regulation of ATR substrate selection by Rad17-dependent loading of Rad9 complexes onto chromatin. Genes Dev 2002;16:198–208.[Abstract/Free Full Text]
  21. Lee J, Kumagai A, Dunphy WG. Claspin, a Chk1-regulatory protein, monitors DNA replication on chromatin independently of RPA, ATR, and Rad17. Mol Cell 2003;11:329–40.[CrossRef][Medline]
  22. Sar F, Lindsey-Boltz LA, Subramanian D, et al. Human claspin is a ring-shaped DNA-binding protein with high affinity to branched DNA structures. J Biol Chem 2004;279:39289–95.[Abstract/Free Full Text]
  23. Bao S, Tibbetts RS, Brumbaugh KM, et al. ATR/ATM-mediated phosphorylation of human Rad17 is required for genotoxic stress responses. Nature 2001;411:969–74.[CrossRef][Medline]
  24. Liu Q, Guntuku S, Cui XS, et al. Chk1 is an essential kinase that is regulated by Atr and required for the G(2)/M DNA damage checkpoint. Genes Dev 2000;14:1448–59.[Abstract/Free Full Text]
  25. Zhao H, Piwnica-Worms H. ATR-mediated checkpoint pathways regulate phosphorylation and activation of human Chk1. Mol Cell Biol 2001;21:4129–39.[Abstract/Free Full Text]
  26. Kim ST, Xu B, Kastan MB. Involvement of the cohesin protein, Smc1, in Atm-dependent and independent responses to DNA damage. Genes Dev 2002;16:560–70.[Abstract/Free Full Text]
  27. Kumagai A, Kim SM, Dunphy WG. Claspin and the activated form of ATR-ATRIP collaborate in the activation of Chk1. J Biol Chem 2004;279:49599–608.[Abstract/Free Full Text]
  28. Shechter D, Costanzo V, Gautier J. ATR and ATM regulate the timing of DNA replication origin firing. Nat Cell Biol 2004;6:648–55.[CrossRef][Medline]
  29. Sorensen CS, Syljuasen RG, Lukas J, Bartek J. ATR, claspin and the Rad9-1-Hus1 complex regulate Chk1 and Cdc25A in the absence of DNA damage. Cell Cycle 2004;3:941–5.[Medline]
  30. Feijoo C, Hall-Jackson C, Wu R, et al. Activation of mammalian Chk1 during DNA replication arrest: a role for Chk1 in the intra-S phase checkpoint monitoring replication origin firing. J Cell Biol 2001;154:913–23.[Abstract/Free Full Text]
  31. Mailand N, Podtelejnikov AV, Groth A, Mann M, Bartek J, Lukas J. Regulation of G2/M events by Cdc25A through phosphorylation-dependent modulation of its stability. EMBO J 2002;21:5911–20.[CrossRef][Medline]
  32. Mailand N, Falck J, Lukas C, et al. Rapid destruction of human Cdc25A in response to DNA damage. Science 2000;288:1425–9.[Abstract/Free Full Text]
  33. Kumagai A, Dunphy WG. Binding of 14-3-3 proteins and nuclear export control the intracellular localization of the mitotic inducer Cdc25. Genes Dev 1999;13:1067–72.[Abstract/Free Full Text]
  34. Yang J, Winkler K, Yoshida M, Kornbluth S. Maintenance of G2 arrest in the Xenopus oocyte: a role for 14-3-3-mediated inhibition of Cdc25 nuclear import. EMBO J 1999;18:2174–83.[CrossRef][Medline]
  35. Lopez-Girona A, Furnari B, Mondesert O, Russell P. Nuclear localization of Cdc25 is regulated by DNA damage and a 14-3-3 protein. Nature 1999;397:172–5.[CrossRef][Medline]
  36. Florensa R, Bachs O, Agell N. ATM/ATR-independent inhibition of cyclin B accumulation in response to hydroxyurea in nontransformed cell lines is altered in tumour cell lines. Oncogene 2003;22:8283–92.[CrossRef][Medline]
  37. Brown EJ, Baltimore D. Essential and dispensable roles of ATR in cell cycle arrest and genome maintenance. Genes Dev 2003;17:615–28.[Abstract/Free Full Text]
  38. Laemmli UK. Claveage of structural protein during the assembly of the head of bacteriophage T4. Nature 1970;227:680–5.[CrossRef][Medline]
  39. Cortez D. Caffeine inhibits checkpoint responses without inhibiting the ataxia-telangiectasia-mutated (ATM) and ATM- and Rad3-related (ATR) protein kinases. J Biol Chem 2003;278:37139–45.[Abstract/Free Full Text]
  40. Sarkaria JN, Tibbetts RS, Busby EC, Kennedy AP, Hill DE, Abraham RT. Inhibition of phosphoinositide 3-kinase related kinases by the radiosensitizing agent wortmannin. Cancer Res 1998;58:4375–82.[Abstract/Free Full Text]
  41. Kashishian A, Douangpanya H, Clark D, et al. DNA-dependent protein kinase inhibitors as drug candidates for the treatment of cancer. Mol Cancer Ther 2003;2:1257–64.[Abstract/Free Full Text]
  42. Lee CM, Fuhrman CB, Planelles V, et al. Phosphatidylinositol 3-kinase inhibition by LY294002 radiosensitizes human cervical cancer cell lines. Clin Cancer Res 2006;12:250–6.[Abstract/Free Full Text]
  43. Xiao Z, Xue J, Sowin TJ, Rosenberg SH, Zhang H. A novel mechanism of checkpoint abrogation conferred by Chk1 downregulation. Oncogene 2005;24:1403–11.[CrossRef][Medline]
  44. Zhou BB, Bartek J. Targeting the checkpoint kinases: chemosensitization versus chemoprotection. Nat Rev Cancer 2004;4:216–25.[CrossRef][Medline]



This article has been cited by other articles:


Home page
 JCBHome page
K. D. Smith, M. A. Fu, and E. J. Brown
Tim-Tipin dysfunction creates an indispensible reliance on the ATR-Chk1 pathway for continued DNA synthesis
J. Cell Biol., October 5, 2009; 187(1): 15 - 23.
[Abstract] [Full Text] [PDF]

Home page
 Genes Dev.Home page
R. Beckerman, A. J. Donner, M. Mattia, M. J. Peart, J. L. Manley, J. M. Espinosa, and C. Prives
A role for Chk1 in blocking transcriptional elongation of p21 RNA during the S-phase checkpoint
Genes & Dev., June 1, 2009; 23(11): 1364 - 1377.
[Abstract] [Full Text] [PDF]

Home page
 J. Biol. Chem.Home page
J.-S. Im and J.-K. Lee
ATR-dependent Activation of p38 MAP Kinase Is Responsible for Apoptotic Cell Death in Cells Depleted of Cdc7
J. Biol. Chem., September 12, 2008; 283(37): 25171 - 25177.
[Abstract] [Full Text] [PDF]

Home page
 Hum Mol GenetHome page
J.-H. Guervilly, G. Mace-Aime, and F. Rosselli
Loss of CHK1 function impedes DNA damage-induced FANCD2 monoubiquitination but normalizes the abnormal G2 arrest in Fanconi anemia
Hum. Mol. Genet., March 1, 2008; 17(5): 679 - 689.
[Abstract] [Full Text] [PDF]

Home page
 Cancer Res.Home page
V. Rodriguez-Bravo, S. Guaita-Esteruelas, N. Salvador, O. Bachs, and N. Agell
Different S/M Checkpoint Responses of Tumor and Non Tumor Cell Lines to DNA Replication Inhibition
Cancer Res., December 15, 2007; 67(24): 11648 - 11656.
[Abstract] [Full Text] [PDF]

Home page
 J. Biol. Chem.Home page
S. Zhang, Y. Zhou, S. Trusa, X. Meng, E. Y. C. Lee, and M. Y. W. T. Lee
A Novel DNA Damage Response: RAPID DEGRADATION OF THE p12 SUBUNIT OF DNA POLYMERASE {delta}
J. Biol. Chem., May 25, 2007; 282(21): 15330 - 15340.
[Abstract] [Full Text] [PDF]

This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow reprints & permissions
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Rodríguez-Bravo, V.
Right arrow Articles by Agell, N.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Rodríguez-Bravo, V.
Right arrow Articles by Agell, N.

HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
Cancer Research Clinical Cancer Research
Cancer Epidemiology Biomarkers & Prevention Molecular Cancer Therapeutics
Molecular Cancer Research Cancer Prevention Research
Cancer Prevention Journals Portal Cancer Reviews Online
Annual Meeting Education Book Meeting Abstracts Online