Cancer Research AACR Membership Sign up for Cancer Research eTOC's
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
Cancer Research 67, 11648, December 15, 2007. doi: 10.1158/0008-5472.CAN-07-3100
© 2007 American Association for Cancer Research
This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Supplementary Data
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.

Molecular Biology, Pathobiology, and Genetics

Different S/M Checkpoint Responses of Tumor and Non–Tumor Cell Lines to DNA Replication Inhibition

Verónica Rodríguez-Bravo, Sandra Guaita-Esteruelas, Noelia Salvador, Oriol Bachs and Neus Agell

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

Requests for reprints: Neus Agell, Departament de Biologia Cellular, Facultat de Medicina, Universitat de Barcelona, C/Casanova, 143, 08036 Barcelona, Spain. Phone: 34-934035267; Fax: 34-934021907; E-mail: neusagell{at}ub.edu.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Cell cycle checkpoint abrogation, especially the inhibition of Chk1 in combination with DNA-damaging treatments, has been proposed as a promising way of sensitizing cancer cells. However, less is known about the possibility to selectively affect tumor cells when they are treated with agents that block DNA synthesis in combination with replication checkpoint inhibitors. Here, we present clear insights in the different responses of tumor and non-transformed cells to the inhibition of DNA replication with hydroxyurea in combination with checkpoint abrogation via inhibition of Ataxia telangiectasia–mutated– (ATM) and Rad3-related/ATM (ATR/ATM) and Chk1 kinases. Interestingly, we find that non-transformed cell lines activate ATR/ATM- and Chk1-independent pathways in response to replication inhibition to prevent mitotic entry with unreplicated DNA. In contrast, tumor cell lines such as HCT116 and HeLa cells rely entirely on Chk1 activity for a proper response to replication inhibitors. Our results show that p38 is activated in response to hydroxyurea treatment and collaborates with Chk1 to prevent mitotic entry in non-transformed cell lines by maintaining cyclin B1/Cdk1 complexes inactive. Furthermore, DNA replication arrest down-regulates cyclin B1 promoter activity in non-transformed cells, but not in tumor cells in a Chk1- and p38-independent way. Thus, our data show that non-transformed cells present a more robust DNA replication checkpoint response compared with tumor cells that involves activation of the p38 pathway. We show that some of these responses to replication block can be lost in tumor cells, causing a defective checkpoint and providing a rationale for tumor-selective effects of combined therapies. [Cancer Res 2007;67(24):11648–56]


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
The maintenance of genomic integrity is essential for the health of multicellular organisms. Consequently, cell cycle checkpoints have evolved to monitor genomic stability and coordinate repair and cell cycle progression (1). Some of those checkpoints are control pathways that delay or prevent cells from entering mitosis under conditions that can compromise genome integrity—for instance, in the presence of damaged or unreplicated DNA. Defects in these processes cause genomic instability and predispose to cancer (2–4). Key proteins involved in monitoring DNA damage are Ataxia telangiectasia–mutated (ATM) and ATM- and Rad3-related (ATR), two serine/threonine kinase members of the phosphoinositide 3-kinase–like family and inhibited by caffeine (5). 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, and apoptosis. Chk1 and Chk2 are key mediator kinases involved in the cell cycle arrest response (2). Regarding the G2-M checkpoint response, these kinases phosphorylate and inactivate Cdc25 phosphatase family (6). Because Cdc25 phosphatase family activates cyclin B1/Cdk1, which is essential for mitotic entry, the cell cycle is finally arrested at the G2-M transition. Apart from this fast response, G2 DNA damage also induces changes in gene expression that finally maintain mitotic entry arrest, such as p53-induced Gadd45, p21, and 14-3-3{sigma} expression or p53-dependent and p53-independent down-regulation of cyclin B1 or Plk1 (7–9).

The family of p38 stress-activated protein kinase (p38) also inhibits mitotic entry in response to diverse events such as osmotic and oxidative stress, UV light, DNA-damaging agents, or microtubule disassembly (10, 11). Once activated, p38 can phosphorylate a variety of cellular targets, including mitogen-activated protein kinase–activated protein kinase-2 (MK2), which, in turn, phosphorylates Cdc25 phosphatases at the same sites as Chk1 and Chk2 (12, 13). The relevance of this pathway in the response to DNA damage varies depending on the cell type, and it has been suggested that it may be an early response to cellular stress. If DNA damage is finally achieved by genotoxic stress, the ATR- and ATM-dependent responses are activated to maintain the inhibition of Cdc25 (14).

To preserve genetic integrity, mammalian cells with stalled replication forks also activate a checkpoint response to delay the initiation of late origins (intra-S checkpoint) and mitotic entry (S/M checkpoint). The exact functioning of this checkpoint is not well understood, but it shares common elements with the one activated by DNA damage. ATR, Rad17, Rad9-Rad1-Hus1 complex and Claspin bind independently to stalled replication forks. Once loaded onto DNA, Rad17 and Claspin collaborate in the activation of ATR that will phosphorylate different substrates as Rad17 itself and Chk1 (15, 16). Chk1 has a central role in the checkpoint response because it finally phosphorylates and inhibits Cdc25 and consequently blocks cyclin B1/Cdk1 activation and mitotic entry when DNA synthesis is inhibited (6, 17). There is increasing evidence that this entire process occurs not only in response to stalled replication forks, but also during the normal S phase, although at a lower intensity, to inhibit mitotic entry while DNA synthesis is still under way (18, 19).

The presence of an ATR- and ATM-independent pathway activated by DNA synthesis inhibition has also been described (20, 21). We have previously described that this pathway leads to a p53-independent down-regulation of cyclin B1 expression (20). Interestingly, this checkpoint response is not functional in most colorectal cancer–derived cell lines analyzed, but is functional in non-transformed cell lines, in HeLa cells, and in derived pancreatic cancer cell lines tested. We have also previously shown that in HeLa cells, Chk1 is essential for the checkpoint response; consequently, in these cells, Chk1 can be activated independently of ATR and ATM (22). Here, we analyze whether in non-transformed cell lines, the ATR- and ATM-independent response needs the participation of Chk1 to avoid mitotic entry when DNA replication is blocked and define the differences with tumor cells. We show that, on the one hand, p38 collaborates with Chk1 to block cyclin B1/Cdk1 activation, and on the other hand, there is an ATR/ATM-, Chk1-, and p38-independent pathway that allows the down-regulation of cyclin B1 expression in non-transformed cell lines. Therefore, our data show that the DNA replication checkpoint has redundant responses to inhibit mitotic entry, and that some of these responses are not functional in tumor cell lines.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Cell culture. HeLa cells, mouse embryo fibroblasts wild-type (MEFs WT), MEFs p38{alpha}–/– (both gift from Dr. Angel Nebreda, Centro Nacional de Investigaciones Oncológicas, Madrid, Spain) and Madin-Darby canine kidney (MDCK) cells were grown in DMEM supplemented with 10% fetal calf serum (FCS). Normal rat kidney (NRK) cells were grown in DMEM supplemented with 5% FCS. HCT116 and SW480 cells were grown in DMEM/HAM F12 (1:1) supplemented with 10% FCS. NIH3T3 cells in DMEM supplemented with 10% donor bovine serum (DBS). NRK, NIH3T3, and HCT116 cells were made quiescent by serum starvation and synchronously activated to proliferate as indicated in Supplementary Methods S1.

Gel electrophoresis and immunoblotting. Cell extracts were obtained and electrophoresed in SDS-polyacrylamide gels and transferred to polyvinylidene difluoride (Millipore) or nitrocellulose (Bio-Rad) membranes (according to the manufacturer's instructions) essentially as previously described (22, 23) and in Supplementary Data S1.

Flow cytometry. To analyze the DNA content, cells were trypsinized, washed with PBS, and centrifuged during 5 min 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 min at room temperature. DNA content was measured by flow cytometry (FACScan Analyser, Becton Dickinson) and analyzed using the Sync Wizard Model, ModFit LT software (Becton Dickinson). For combined analysis of DNA content and bromodeoxyuridine (BrdUrd) incorporation, cells were first incubated for 30 min with 10 µmol/L of BrdUrd analyzed using the FITC BrdUrd Flow Kit (BD PharMingen) following the manufacturer's instructions.

Immunocytochemistry and laser scanning cytometry analysis. Quantification of the percentage of cells positive for phospho-histone H3 (Ser10) by laser scanning cytometry (LSC) was done exactly as previously described (22). To detect phospho-H2AX (P-H2AX) foci, immunocytochemistry was done as for phospho-H3, but using primary antibody against P-H2AX (Upstate Biotechnology Inc.; 1:200 dilution).

Lamina B, {alpha}-tubulin, and cyclin B1 were analyzed by immunocytochemistry using antibodies against Lamina B (Santa Cruz Biotechnology M-20; 1:50 dilution), {alpha}-tubulin FITC or cyclin B1 (Upstate Biotechnology Inc.; 1:20 dilution) as indicated in Supplementary Data S1. BrdUrd immunocytochemistry was done as previously described (24) after incubation of the cells with BrdUrd (10 µmol/L).

Small interfering RNA. NIH3T3 cells were grown for 1 day on glass coverslips and transfected with either the mouse specific small interfering RNA (siRNA) SMARTpools of Chk1 (Dharmacon) or the p38{alpha} and p38β siRNA SMARTpools (MAPK14 and MAPK11, both Dharmacon), using LipofectAMINE 2000 (Invitrogen) following the manufacturer's instructions. For HeLa cells, the procedure was the same, but using human specific siRNA SMARTpool of Chk1 (Dharmacon; ref. 22). Control experiments were done transfecting the cells with a green fluorescent protein (GFP) duplex siRNA (NNGGCUACGUCCAGGAGCGCA sequence, Dharmacon).

Cyclin B1 promoter activity regulation. NIH3T3 cells were co-transfected with 150 ng of pGL3-CycB1 promoter (–872 to +22, upstream of a luciferase reporter gene; gift of Dr. K.-M. Yao, Universiy of Hong Kong) and 10 ng of pRLSV-Luc. In all experiments, luciferase activity was assayed 48 h after transfection and normalized taking into account the value obtained with Renilla luciferase. Firefly luciferase (Luc) and Renilla luciferase activities were measured using the Dual Luciferase Reporter Assay System (Promega) according to the manufacturer's instructions. Duplicates or triplicates were systematically included, and experiments were repeated at least thrice.

Colony formation assay. HCT116 and NRK synchronized cells were treated in the S phase with 1.5 mmol/L hydroxyurea, 5 mmol/L caffeine (Sigma), 300 nmol/L UCN-01 (National Cancer Institute), and different combinations of these inhibitors. After 6 h, cells were trypsinized, counted, and seeded in 60-mm dishes (1,000 cells per dish). After this, cells were kept growing in fresh medium without any drug during 10 days. After this time, colonies were fixed, stained with crystal violet solution, and counted.

Immunoprecipitation and Cdc2 kinase assay. Cells were lysed for 30 min at 4°C in IP buffer [50 mmol/L Tris-HCl (pH 7.4), 0.1% Triton X-100, 5 mmol/L EDTA, 250 mmol/L NaCl, 1 mmol/L NaF, 0.1 mmol/L Na3VO4, 1 mmol/L phenylmethylsulfonyl fluoride, 1 µg/mL aprotinin, and 10 µg/mL leupeptin]. Lysates were clarified by centrifuging at 10,000 x g for 10 min. Lysates (500 µg of protein for NRK cells or 100 µg for HCT116 cells) were incubated with 2 µg of anti-cyclin B1 (Upstate Biotechnology) antibodies or with 2 µg of mouse immunoglobulin Gs (IgG) as a control overnight at 4°C. Protein immunocomplexes were pulled down with G-Sepharose (Sigma) and kinase activity analyzed exactly as described previously (20). Data were recorded using Personal FX (Bio-Rad) and analyzed with Quantity One software (Bio-Rad).


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
ATR/ATM and Chk1 inhibition does not abrogate hydroxyurea-induced S/M response in non-transformed cell lines. Most tumor-derived cell lines have a checkpoint response to DNA synthesis inhibition that relies entirely on Chk1 and is consequently abrogated by UCN01. We have previously shown that non-transformed cell lines such as NRK cells, NIH3T3, or MEFs have a caffeine-insensitive checkpoint response (20). Because caffeine inhibits ATR and ATM, kinases that can activate Chk1, here, we tested whether the checkpoint response to hydroxyurea treatment described in non-transformed cell lines is dependent on this kinase. HeLa and HCT116 were used as tumor cell lines, both of epithelial origin. NRK, NIH3T3, and MDCK, the first two fibroblastic and the last epithelial, were used as non–tumor cell lines. To activate the DNA replication checkpoint response, asynchronous cells were treated with 1.5 mmol/L hydroxyurea for 25 h. The functionality of the checkpoint was determined analyzing the percentage of phospho-histone H3 (P-H3)–positive cells. As shown in Fig. 1A , all non-transformed cell lines analyzed had a functional checkpoint response because all of them inhibited mitotic entry when treated with hydroxyurea. In agreement with our previous results, addition of caffeine during the last 10 h abrogated the checkpoint response in HCT116 cells, but not in the other cell lines analyzed. Furthermore, as in previous research, the checkpoint response in HeLa and HCT116 cells was abrogated by UCN01, a Chk1 inhibitor. UCN01 also abrogated checkpoint response in SW480 and HT29 colorectal cancer tumor cells (data not shown). In contrast, UCN01 did not abrogate the checkpoint response in either NRK, NIH3T3, or MDCK cells. Therefore, all non-transformed cell lines analyzed here, either fibroblastic or epithelial, had a UCN01-insensitive checkpoint response that was not functional in tumor-derived cell lines. Moreover, neither the checkpoint was abrogated when using 20 µmol/L SB218078 (Sigma), another unrelated Chk1 inhibitor. To finally prove Chk1 participation, we knocked down Chk1 expression using a siRNA pool specific for Chk1. As shown in Fig. 1B, siRNA inhibited Chk1 expression, but NIH3T3 cells did not enter mitosis in the presence of hydroxyurea. In contrast, and confirming our previously reported data (22), hydroxyurea-treated HeLa cells entered mitosis when transfected with a siRNA pool specific for Chk1.


Figure 1
View larger version (34K):
[in this window]
[in a new window]
[Download PPT slide]
  		Figure 1. Different effects of Chk1 inhibition and knockdown on the hydroxyurea-induced checkpoint response in non-tranformed cell lines and tumor cell lines. A, the different asynchronous cell lines were treated, where indicated, with 1.5 mmol/L hydroxyurea (HU) for 25 h. During the last 10 h of treatment, 5 mmol/L caffeine (Caff), 300 nmol/L UCN01 (UCN), 10 µmol/L SB218078 (218078), or a combination of those drugs was added to the culture media. After treatment, cells were fixed, and immunocytochemistry and LSC analysis were done to determine the percentage of P-H3–positive cells as indicated in Materials and Methods. Representative experiments are shown (top graph, non-transformed cell lines; bottom graph, tumor cell lines). B, asynchronous NIH3T3 cells were transfected with siRNAs to knock down Chk1 (Chk1i) or GFP as a control (GFPi). Forty-eight hours after transfection, the levels of Chk1 and actin were analyzed by Western blot (top left for NIH3T3 cells). At that time, cells were also treated for 25 h with 1.5 mmol/L HU or nothing (–), and during the last 10 h, where indicated, 5 mmol/L caffeine was added (Caff). Top, microscope images of phospho-H3 (P-H3) immunostaining and propidium iodine (PI) staining of NIH3T3 cells are shown. Bottom, graph showing the quantification of a representative experiment with NIH3T3 cells. Also included are HeLa cells as a positive control (22).

 
To avoid excessive exposure to hydroxyurea, which might have caused DNA damage, cells were synchronized in G0 and released into high serum as indicated in Supplementary Methods S1. Once in the S phase (16 h after serum addition for NRK cells), checkpoint activation was induced by 6 h of hydroxyurea treatment, and then the percent of P-H3 was analyzed. Treatment of these cells with hydroxyurea for 6 h completely abolished DNA replication because DNA content did not increase during this time (Supplementary Data S2). NRK cells responded to hydroxyurea treatment, activating the checkpoint response correctly and consequently inhibiting mitotic entry (measured as P-H3–positive cells). As with asynchronous populations, UCN01 did not abrogate DNA replication checkpoint activation in non-transformed cell lines (Fig. 2A ). The lack of formation of P-H2AX foci upon hydroxyurea treatment indicated that no extensive DNA damage was produced either when treating asynchronously growing cells with hydroxyurea for 24 h or S-phase cells with hydroxyurea for 6 h (Fig. 2B). The same results were obtained with NIH3T3 cells (data not shown). To ensure that hydroxyurea did not induce an irreversible arrest in non-transformed cell lines, we confirmed that NRK cells could re-enter the cell cycle upon hydroxyurea removal because they synthesized DNA (6 h after hydroxyurea removal, more than 90% of the cells incorporated BrdUrd) and entered mitosis (determined by H3 phosphorylation; Fig. 2C).


Figure 2
View larger version (43K):
[in this window]
[in a new window]
[Download PPT slide]
  		Figure 2. UCN01 does not abrogate checkpoint responses to HU in synchronized NRK cells. A, S-phase synchronized NRK cells (16 h after activation by serum addition) were treated for 6 h with the specified drugs. Cells were fixed at the indicated time after activation, and the percentage of P-H3–positive cells was analyzed by immunocytochemistry and quantified by LSC. B, S synchronized NRK cells (left) and asynchronously growing NRK cells (right) were treated for 6 or 24 h with the different drugs and immunostained to detect P-H2AX foci. C, S-phase synchronized NRK cells were treated with hydroxyurea for 6 h, and then the medium was changed, and BrdUrd was added. At the times indicated after hydroxyurea removal, cells were fixed and processed for immunocytochemistry to detect P-H3 or incorporated BrdUrd. D, S-phase synchronized NRK cells or S-phase synchronized HCT116 were treated for 6 h with the drugs. Viability was then determined by clonogenic assay. Top, quantification of three different experiments; bottom, colonies obtained in a representative experiment. HU, 1.5 mmol/L hydroxyurea; Caff, 5 mmol/L caffeine; UCN, 300 nmol/L UCN01; Adr, 0.3 µg/mL Adriamycin.

 
In addition, the functionality of the hydroxyurea-induced checkpoint response in the presence of either caffeine or Chk1 inhibitors was directly analyzed by testing the survival of the cells using colony formation assays. S-phase NRK and HCT116 cells were treated for 6 h with hydroxyurea to activate the checkpoint response in the presence or absence of caffeine or UCN01. The inhibitors were then washed out, and colony assays were done. As shown in Fig. 2D, NRK cells had high viability after combined treatment with DNA synthesis inhibitors plus caffeine or UCN01 (or even both together), confirming the presence of a Chk1- and ATR/ATM-independent checkpoint response to DNA synthesis inhibition in these cells that was not functional in the tumor cell line HCT116.

Decrease in cyclin B1 accumulation after hydroxyurea treatment is ATR/ATM and Chk1 independent in non-transformed cell lines. We have previously shown that in cells with a functional ATR/ATM-independent response to DNA synthesis inhibition, such as NRK or NIH3T3, hydroxyurea treatment inhibited the normal accumulation of cyclin B1 observed from S phase to mitosis. This response was p53 independent because p53–/– cells showed the same behavior (20). In contrast, in cells whose caffeine-insensitive checkpoint response was not functional, like HCT116 cells, cyclin B1 continued to accumulate when DNA synthesis was inhibited. We now tested whether this effect on cyclin B1 accumulation induced by hydroxyurea treatment was Chk1 independent in NRK cells. When S-phase NRK cells were treated for 6 h with hydroxyurea, both Chk1 activation (determined by its phosphorylation in Ser345) and inhibition of cyclin B1 accumulation were observed (Fig. 3A ). Although the combined hydroxyurea and caffeine treatment did not induce Chk1 phosphorylation, cyclin B1 levels were still low. Consequently, hydroxyurea-induced inhibition of cyclin B1 accumulation and stop of mitotic entry may occur in the absence of Chk1 phosphorylation at Ser345. Furthermore, Chk1 activity was not needed to inhibit cyclin B1 accumulation in response to hydroxyurea treatment because UCN01 addition did not abrogate the effect of hydroxyurea on cyclin B1 levels (Fig. 3A). Similar results were also obtained using synchronized NIH3T3 cells (Fig. 3B).


Figure 3
View larger version (40K):
[in this window]
[in a new window]
[Download PPT slide]
  		Figure 3. Chk1 phosphorylation and activity are dispensable for the inhibition of cyclin B1 expression observed upon hydroxyurea treatment in NRK and NIH3T3 cells. S-phase synchronized (16 h after serum addition) NRK cells (A) or NIH3T3 cells (B) were treated for 6 h with the specified drugs. Cell lysates were then processed at the indicated times after serum addition to analyze by Western blotting the levels of cyclin B1 (CycB1), Chk1, P-Chk1Ser345, and actin as a loading control. HU, 1.5 mmol/L hydroxyurea; Caff, 5 mmol/L caffeine; UCN, 300 nmol/L UCN01.

 
p38{alpha}/β collaborates with Chk1 in the S/M checkpoint response to hydroxyurea treatment. Participation of p38{alpha} and p38β in parallel to Chk1 and Chk2 in the response to DNA damage has already been described (12, 13, 25). Moreover, we have previously shown that hydroxyurea treatment of S-phase synchronized NRK cells induced activation of p38 (20). However, the participation of p38 in the response to DNA replication block in the absence of an extensive DNA damage has not been thoroughly investigated. Therefore, we tested whether p38 participated in the Chk1-independent checkpoint response to DNA synthesis inhibition in NRK and NIH3T3. To this end, S-phase synchronized NRK and NIH3T3 cells were treated with hydroxyurea plus caffeine, UCN01, or SB202190 (Sigma; an inhibitor of p38{alpha} and p38β) alone or in combination, and the percentage of P-H3–positive cells was analyzed. As shown in Fig. 4A , none of the drugs on their own abrogated the checkpoint response to hydroxyurea treatment (functionality of p38 inhibitor is shown in Supplementary Data S3). However, the combination of UCN01 plus SB202190 disrupted the normal S/M checkpoint response to hydroxyurea because after this treatment, the percentage of P-H3–positive cells increased to the levels observed in nontreated cells. Of course, those P-H3–positive cells presented abnormal mitosis because DNA was condensing before being fully replicated (Supplementary Data S4). Interestingly, caffeine plus SB202190 treatment in the presence of hydroxyurea did not abrogate the checkpoint, supporting the finding that Chk1 can be activated independently of ATR/ATM activity (22). The same results were obtained with another nonrelated p38{alpha}/β inhibitor, SB203580 (Sigma; Supplementary Data S5). Consequently, these results showed that the simultaneous inhibition of p38 and Chk1 in non-transformed cells was needed to abrogate the correct S/M replication checkpoint response to hydroxyurea, indicating a collaboration of Chk1 and p38{alpha}/β in this checkpoint response.


Figure 4
View larger version (28K):
[in this window]
[in a new window]
[Download PPT slide]
  		Figure 4. Simultaneous inhibition of p38 and Chk1 abrogates the checkpoint response to hydroxyurea in NRK and NIH3T3 cells. A, S-phase synchronized (16 h after serum addition) NRK and NIH3T3 cells were treated for 6 h with the specified drugs, and at the indicated times after serum addition, cells were fixed, and the percentage of P-H3–positive cells was analyzed by immunocytochemistry and quantified by LSC. B, left, S-phase synchronized HCT116 (12 h after serum addition) were treated for 6 h with the specified drugs and at the indicated time after serum addition processed and analyzed as in A. Right, asynchronously growing HeLa cells were treated (HU) or not (–) for 25 h with hydroxyurea, and where indicated, SB202190 (SB) was added during the last 10 h. C, cells were treated (HU) or not (–) for 25 h with hydroxyurea. They were then lysed, and phosphorylation of p38 (P-p38) was analyzed by Western blotting. Actin was also analyzed as loading control. HU, 1.5 mmol/L hydroxyurea; Caff, 5 mmol/L caffeine; UCN, 300 nmol/L UCN01; SB, 20 µmol/L SB202190.

 
In tumor cell lines (HCT116 and HeLa cells), UCN01 treatment abrogated the checkpoint response (ref. 22 and Fig. 1A), but, as in non-transformed cell lines, inhibition of p38 alone (SB202190 addition) did not (Fig. 4B).

Because p38 was participating in the cell cycle arrest induced by hydroxyurea treatment in non-transformed but not in tumor cell lines, phosphorylation of p38 measured upon hydroxyurea treatment was analyzed. As shown in Fig. 4C, in all cell lines analyzed, either tumor or non-transformed cell lines, hydroxyurea treatment induced p38 phosphorylation. Consequently, the lack of functionality of the p38 pathway in these tumor cell lines was not due to the lack of p38 activation.

To further confirm p38 participation in the DNA replication checkpoint in non-transformed cell lines, MEFs knocked out for p38{alpha} were used (Fig. 5A ). These p38{alpha} knocked out cells still had a checkpoint response to hydroxyurea treatment even in the presence of UCN01, but it was abrogated when UCN01 and SB202190 were simultaneously added. The fact that p38 phosphorylation (indicative of its activity) and MK2 phosphorylation (a substrate of p38 kinases) were induced by hydroxyurea in cells lacking p38{alpha} indicated that p38β could probably be involved in the checkpoint response (Fig. 5A and B). To finally prove the collaboration between p38 and Chk1 in the hydroxyurea-induced checkpoint response, p38{alpha} and p38β were knocked down in NIH3T3 cells using siRNA. As shown in Fig. 5C, simultaneous knockdown of both p38{alpha} and p38β abrogated the checkpoint response to hydroxyurea only when Chk1 was inhibited by UCN01 at the same time. Consistent with this idea, knocking down Chk1 by transfecting specific siRNA allowed hydroxyurea-treated cells to enter mitosis only in the presence of the p38{alpha}/β inhibitor SB202190. To prove that p38{alpha} and p38β siRNA treatment was in fact knocking down any isoform of p38 that could be activated by hydroxyurea treatment, phosphorylation of p38 was analyzed by Western blotting (Fig. 5C). Only when p38 was not phosphorylated could UCN01 treatment abrogate the S/M checkpoint response to hydroxyurea. All these results confirmed a collaboration between p38{alpha}/β and Chk1 to inhibit mitotic entry in response to DNA synthesis inhibition.


Figure 5
View larger version (26K):
[in this window]
[in a new window]
[Download PPT slide]
  		Figure 5. Effect of p38 knockdown on the checkpoint response to DNA replication inhibition in non-transformed cell lines. A, asynchronously growing WT and p38{alpha}–/– immortalized MEFs were treated where indicated for 25 h with 1.5 mmol/L hydroxyurea (HU). Also, where indicated, 300 nmol/L UCN01 (UCN), 20 µmol/L SB202190 (SB), or a combination of these two drugs was added during the last 10 h of hydroxyurea treatment. Cells were processed for immunocytochemistry and LSC to quantify the percentage of cells P-H3 positive (top) or lysed to determine the amount of p38{alpha} by Western blotting, or the levels of MK2 phosphorylated at Thr334 (P-MK2; bottom). Actin was analyzed as loading control. A representative experiment out of three is shown. B, asynchronously growing WT and p38{alpha}–/– immortalized MEFs were treated where indicated for 25 h with 1.5 mmol/L hydroxyurea. Where indicated, hydroxyurea or 300 nmol/L UCN01 was added to the media for the last 10 h. Cells were lysed and processed to analyze the presence of p38{alpha} and p38 phosphorylation (P-p38) by Western blotting. A representative experiment out of three is shown. C, NIH3T3 cells were transfected with siRNAs to knock down Chk1 (Chk1i), p38{alpha} and p38β (p38i), or GFP (GFPi) or non-transfected (–). Cells were then serum starved and synchronously induced to re-enter the cell cycle. Once in S phase (16 h after serum addition), cells were treated for 6 h with 1.5 mmol/L hydroxyurea, 300 nmol/L UCN01, and 20 µmol/L SB202190 at the indicated combination. Cells were then processed for immunocytochemistry and LSC to quantify the percentage of cells P-H3 positive (graph) or lysed and processed to analyze the presence of Chk, and p38 phosphorylation by Western blotting. GAP120 Western blot was done as a control of loading. A representative Western blot is shown.

 
Chk1 and p38 collaborate to inhibit CyclinB1/Cdk1 activity upon hydroxyurea treatment but are not essential to down-regulate cyclin B1 levels. Unexpectedly, although hydroxyurea-treated cells were entering mitosis upon UCN01 plus SB202190 addition, cyclin B1 levels did not recover in response to this treatment (Fig. 6A ). Thus, although they entered mitosis, S-phase NRK cells (16 h) treated with hydroxyurea plus UCN01 and SB202190 for 6 h presented the same low levels of cyclin B1 observed before treatment at 16 h. Analyzing cyclin B1/Cdk1 activity in those cells, we saw that hydroxyurea treatment inhibited the activation of this complex. Although further addition of UCN01 or SB202190 alone in the presence of hydroxyurea did not recover activity, it was partially recovered when a combination of the two inhibitors was added (Fig. 6B). In contrast, and confirming the fact that Chk1 inhibition abrogated the checkpoint response in HCT116 cells, UCN01 treatment alone recovered cyclin B1–associated kinase activity of hydroxyurea-treated HCT116 cells (Fig. 6B). Recovery of cyclin B1 activity observed in NRK by the addition of UCN01 and SB202190 was not complete, but it was stronger if we compare cyclin B1 kinase–associated activity to the total amount of cyclin B1 in each condition (Fig. 6C). Moreover, most P-H3–positive NRK cells observed in UCN01, SB202190, and hydroxyurea-treated cells also had disrupted nuclear lamina, indicating that Cdk1 was in fact active (Supplementary Data S6A). Furthermore, nuclear localization of cyclin B1 was only observed in control conditions and upon simultaneous inhibition of Chk1 and p38{alpha}/β (Supplementary Data S6B). We conclude then that in NRK cells, Chk1 and p38 work in parallel to inhibit cyclin B1/Cdk1 complex nuclear accumulation and activation, but that some other factor is responsible for the down-regulation of cyclin B1 expression. In cells treated with hydroxyurea during the S phase, the amount of cyclin B1 present in each cell may have been enough to allow Cdk1 activation and, consequently, mitotic entry, when Chk1 and p38{alpha}/β were inhibited. To prove this, hydroxyurea was added to NRK cells 10 h after activation from quiescence, when most cells had not yet entered S phase and consequently had undetectable levels of cyclin B1. In this case hydroxyurea inhibited cell cycle just at the beginning of the S phase (G1-S transition, 2n DNA content), and posterior addition of UCN01 plus SB202190 did not abrogate the checkpoint response (Supplementary Data S7 for NRK and data not shown for NIH3T3 cells).


Figure 6
View larger version (36K):
[in this window]
[in a new window]
[Download PPT slide]
  		Figure 6. Cyclin B1 protein levels, kinase-associated activity, and promoter activity upon activation of the DNA replication checkpoint in the presence of UCN01 and SB202190. S-phase synchronized NRK cells (16 h after serum addition) were treated with the specified drugs for 6 h. A, top and bottom, at the indicated time after serum addition, cells were lysed and processed to analyze levels of cyclin B1 (CycB1) and actin by Western blotting. A representative experiment out of three is shown. B, cells were processed for immunoprecipitation (IP) to analyze cyclin B1 (anti-CycB1)–associated H1 kinase activity. Nonrelated mouse IgG was used as immunoprecipitation control (mIgG). A representative experiment out of three is shown. Bottom, cyclin B1–associated kinase activity of HCT116-synchronized cells after the same treatments is shown. C, the ratio cyclin B1-associated H1 kinase activity of NRK cells versus total levels of cyclin B1 from three different experiments, as in A and B, was determined. The graph shows the variation of this ratio with respect to the value obtained in mitotic cells (22 h after activation; 100% value). D, asynchronously growing NIH3T3 cells were co-transfected with pGL3-CycB1 promoter (–872 to +22, upstream of a luciferase reporter gene) and pRLSV-Luc and, 32 h later, where indicated, treated with hydroxyurea for 16 h more. During the last 6 h, the indicated drugs were added. HU, 1.5 mmol/L hydroxyurea; Caff, 5 mmol/L caffeine; UCN, 300 nmol/L UCN01; SB, 20 µmol/L SB202190.

 
We have previously shown that low levels of cyclin B1 upon hydroxyurea treatment correlated with a decrease in cyclin B1 mRNA levels. We now aimed to analyze whether this occurred in parallel to a decrease in promoter activity, and whether, as expected from the above results, this regulation was independent of Chk1 and p38 activity. As shown in Fig. 6D, hydroxyurea treatment inhibited cyclin B1 promoter activity by more than 50%, and neither caffeine, UCN01, nor SB202190, whether alone or in combination, was able to reverse the effect. This indicated that ATR/ATM, Chk1, and p38 were dispensable for down-regulating cyclin B1 promoter activity upon DNA synthesis inhibition. Interestingly, in HCT116 cells, which do not show a decrease in cyclin B1 protein levels with hydroxyurea treatment (20), cyclin B1 promoter activity was not down-regulated by hydroxyurea treatment (Supplementary Data S8).


   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
Here we analyze the response of non-transformed cell lines to DNA synthesis inhibition induced by hydroxyurea treatment. Our data indicate that in non-transformed mammalian cell lines, the checkpoint response is more complex and robust than has been shown up to now using tumor cell lines. Although in tumor cell lines (2, 17), embryonic stem cells (26–28), and nonmammalian cells (29, 30), Chk1 has been reported to be an essential kinase for the DNA replication checkpoint, in all non-transformed mammal cell lines used in this study, additional parallel pathways were found that ensure replication checkpoint response when Chk1 and ATR/ATM are not functional.

Most checkpoints activate non-transcriptional fast responses and also delayed transcriptional checkpoint responses that are needed to maintain a longer effect (7, 31). Results presented in this paper, together with our previous results, show that upon DNA replication inhibition, there are transcriptional and non-transcriptional ATR/ATM- and Chk1-independent responses. When DNA synthesis is inhibited in NRK or NIH3T3 cells, we observe features that indicate cell cycle arrest and, consequently, mitotic entry inhibition (activation of S/M checkpoint): lack of cyclin B1 accumulation, lack of nuclear translocation of the preexisting cyclin B1, inhibition of cyclin B1/Cdk1 activation, inhibition of lamina disaggregation, inhibition of H3 phosphorylation, and lack of chromatin condensation. None of these features were abrogated by the exclusive inhibition of Chk1 or ATR and ATM. In contrast, Chk1 inhibition abrogated all these S/M checkpoint responses in all tumor cell lines analyzed here. However, the dependence of the intra-S checkpoint response upon DNA inhibition on Chk1 has not been analyzed here.

We analyzed here whether the p38 stress-activated kinase, which participates in DNA damage and osmotic stress cellular responses (10, 12–14, 25, 32), may also participate in the cellular response to DNA synthesis inhibition. Inhibition of p38 alone did not abrogate any of these checkpoint responses, but simultaneous inhibition of both Chk1 and p38 allowed cells treated with hydroxyurea in S phase to enter mitosis. Although cyclin B1 protein levels did not increase, we observed an increase in cyclin B1–associated kinase activity when compared with hydroxyurea-treated cells. It is well known that when DNA replication is inhibited, Chk1 activation induces phosphorylation and inhibition of the Cdc25 phosphatase family (6). Consequently, cyclin B1/Cdk1 complexes cannot be activated, and mitotic entry is inhibited. Our data indicate that in non-transformed cell lines, p38{alpha}/β and Chk1 collaborate to prevent cyclin B1–associated kinase activity upon DNA synthesis inhibition. It has previously been shown that MK2, one of the p38 substrates, is activated upon osmotic stress and UV light irradiation and, in response to DNA-damaging agents, inhibits Cdc25 (11, 12, 14, 25). Here, we show that hydroxyurea addition induces p38 and MK2 phosphorylation. Consequently, p38/MK2 might collaborate with Chk1 to inhibit the activation of Cdc25 and, thus, cyclin B1/Cdk1 in response to hydroxyurea. However, p38 may inhibit cyclin B1–associated kinase activity via other mechanisms. Reinhardt et al. (25), using MEF p53–/– or U2OS cells, found that upon DNA damage, the inhibition of MK2 alone is able to abrogate the DNA damage checkpoint response. Those results would agree with ours if MK2 could be activated also by Chk1.

Another open question is how DNA synthesis inhibition activates p38. Although activation of p38 by DNA damaging agents has been shown to be ATM/ATR dependent (25, 33) and correlates with the appearance of P-H2AX foci, our data indicate that p38 is activated by hydroxyurea even in the presence of caffeine and in the absence of these P-H2AX foci. Changes in chromatin topology, induced, for instance, by topoisomerase II and histone deacetylase inhibitors, also delay G2-M transition by triggering the p38 checkpoint pathway (34). It is possible that DNA synthesis inhibition also induces chromatin topology changes, leading to the activation of p38 in parallel to ATR and Chk1.

We also show here the existence of a second Chk1-independent response, which leads to cyclin B1 promoter down-regulation. Our data indicate that, in non-transformed fibroblasts, DNA synthesis inhibition induces a decrease in cyclin B1 promoter activity that is not reversed by caffeine addition, Chk1, or p38 inhibition, either alone or in combination. This response would only be important to inhibit mitosis entry in cells that have been arrested at the very beginning of the S phase, when the cyclin B1 levels have not yet increased, because, as indicated above, low levels of cyclin B1 may allow mitosis entry. Transcriptional regulation of cyclin B1 gene (8, 35, 36), as well as regulation of cyclin B1 mRNA stability (37) during the cell cycle, has been reported. We have also previously shown that Claspin down-regulates cyclin B1 expression in HeLa cells (22). Further investigation is needed to elucidate the link between DNA synthesis inhibition and cyclin B1 promoter activity, but interestingly, this is a checkpoint response that is not functional in some tumor cell lines (20).

Because checkpoint signaling pathways facilitate cell survival following DNA damage in human cells, there is great interest in combining checkpoint inhibitors with genotoxic anticancer treatments as a means to enhance tumor susceptibility. In fact, it has been reported in several preclinical and clinical studies that inhibition of Chk1 enhances the sensitivity of tumor cells to chemotherapy (38). We have shown here that although Chk1 is an essential DNA replication checkpoint kinase in tumor cell lines, it is not essential in non-transformed cell lines. Consequently, Chk1 inhibition drastically enhances the cytotoxic effect of hydroxyurea treatment in HCT116 cells, as determined by clonogenic assays, whereas hardly any effect was observed in NRK cells. Blocking DNA replication has been a major target for anticancer therapy, and new ribonucleotide reductase inhibitors have been tested and applied in clinical practice (39). Among the different types of ribonucleotide reductase inhibitors developed at the moment, some are nucleoside analogues that also induce DNA damage because they are incorporated into DNA as, for instance, 5-fluorouracil. In this study, we have used hydroxyurea to activate replication checkpoint. Hydroxyurea is a radical scavenger that inactivates ribonucleotide reductase, and because it does not incorporate into DNA, DNA damage induced by hydroxyurea is lower. Although clinical application of hydroxyurea is reduced, other radical scavengers have been developed and are in phase I and II clinical trials (for example, Didox and Tridox). In conclusion, our data showing that nontumor cells have Chk1-independent checkpoint responses to DNA replication inhibition that are not functional in tumor cells provide a rationale for tumor-selective effects of combined therapies.


   Acknowledgments
 
Grant support: SAF2001-2901, SAF2004-02159, and GEN2003-20243-C08-01. Verónica Rodríguez is the recipient of a predoctoral fellowship from Consell Interdepartamental de Recerca i Innovació Tecnològica. Noelia Salvador is the recipient of a predoctoral fellowship from the Spanish Government. Sandra Guaita-Esteruelas has a postdoctoral contract from Instituto de Salut Carlos III (FIS).

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.

We thank Dr. Angel Nebreda (Centro Nacional de Investigaciones Oncológicas, Madrid) for providing us with p38{alpha}–/– cells.


   Footnotes
 
Note: Supplementary data for this article are available at Cancer Research Online (http://cancerres.aacrjournals.org/).

Received 8/13/07. Revised 10/ 9/07. Accepted 10/16/07.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

  1. Hartwell LH, Weinert TA. Checkpoints—controls that ensure the order of cell-cycle events. Science 1989;246:629–34.[Abstract/Free Full Text]
  2. Bartek J, Lukas J. Chk1 and Chk2 kinases in checkpoint control and cancer. Cancer Cell 2003;3:421–9.[CrossRef][Medline]
  3. Kastan MB, Bartek J. Cell-cycle checkpoints and cancer. Nature 2004;432:316–23.[CrossRef][Medline]
  4. Cahill DP, Lengauer C, Yu J, et al. Mutations of mitotic checkpoint genes in human cancers. Nature 1998;392:300–3.[CrossRef][Medline]
  5. Shiloh Y. ATM and related protein kinases: safeguarding genome integrity. Nat Rev Cancer 2003;3:155–68.[CrossRef][Medline]
  6. Mailand N, Podtelejnikov AV, Groth A, et al. Regulation of G2/M events by Cdc25A through phosphorylation-dependent modulation of its stability. EMBO J 2002;21:5911–20.[CrossRef][Medline]
  7. Taylor WR, Stark GR. Regulation of the G2/M transition by p53. Oncogene 2001;20:1803–15.[CrossRef][Medline]
  8. Crawford DF, Piwnica-Worms H. The G2 DNA damage checkpoint delays expression of genes encoding mitotic regulators. J Biol Chem 2001;276:37166–77.[Abstract/Free Full Text]
  9. Levesque AA, Kohn EA, Bresnick E, et al. Distinct roles for p53 transactivation and repression in preventing UCN-01–mediated abrogation of DNA damage-induced arrest at S and G(2) cell cycle checkpoints. Oncogene 2005;24:3786–96.[CrossRef][Medline]
  10. Bulavin DV, Higashimoto Y, Popoff IJ, et al. Initiation of a G2/M checkpoint after ultraviolet radiation requires p38 kinase. Nature 2001;411:102–7.[CrossRef][Medline]
  11. Lemaire M, Froment C, Boutros R, et al. CDC25B phosphorylation by p38 and MK-2. Cell Cycle 2006;5:1649–53.[Medline]
  12. Lopez-Aviles S, Grande M, Gonzalez M, et al. Inactivation of the Cdc25 phosphatase by the stress-activated Srk1 kinase in fission yeast. Mol Cell 2005;17:49–59.[CrossRef][Medline]
  13. Manke IA, Nguyen A, Lim D, et al. MAPKAP kinase-2 is a cell cycle checkpoint kinase that regulates the G(2)/M transition and S phase progression in response to UV irradiation. Mol Cell 2005;17:37–48.[CrossRef][Medline]
  14. Bulavin DV, Amundson SA, Fornace J. p38 and Chk1 kinases: different conductors for the G2/M checkpoint symphony. Curr Opin Genet Dev 2002;12:92–7.[CrossRef][Medline]
  15. 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]
  16. 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]
  17. 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]
  18. 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]
  19. Sorensen CS, Syljuasen RG, Lukas J, et al. 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]
  20. 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]
  21. 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]
  22. Rodriguez-Bravo V, Guaita-Esteruelas S, Florensa R, et al. Chk1- and claspin-dependent but ATR/ATM- and Rad17-independent DNA replication checkpoint response in HeLa cells. Cancer Res 2006;66:8672–9.[Abstract/Free Full Text]
  23. Laemmli UK. Cleavage of structural protein during the assembly of the head of bacteriophage T4. Nature 1970;227:680–5.[CrossRef][Medline]
  24. Rodriguez-Vilarrupla A, Diaz C, Canela N, et al. Identification of the nuclear localization signal of p21(cip1) and consequences of its mutation on cell proliferation. FEBS Lett 2002;531:319–23.[CrossRef][Medline]
  25. Reinhardt HC, Aslanian AS, Lees JA, et al. p53-deficient cells rely on ATM- and ATR-mediated checkpoint signaling through the p38MAPK/MK2 pathway for survival after DNA damage. Cancer Cell 2007;11:175–89.[CrossRef][Medline]
  26. Niida H, Tsuge S, Katsuno Y, et al. Depletion of Chk1 leads to premature activation of Cdc2-cyclin B and mitotic catastrophe. J Biol Chem 2005;280:39246–52.[Abstract/Free Full Text]
  27. 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]
  28. Takai H, Tominaga K, Motoyama N, et al. Aberrant cell cycle checkpoint function and early embryonic death in Chk1(–/–) mice. Genes Dev 2000;14:1439–47.[Abstract/Free Full Text]
  29. Guo ZJ, Kumagai A, Wang SX, et al. Requirement for Atr in phosphorylation of Chk1 and cell cycle regulation in response to DNA replication blocks and UV-damaged DNA in Xenopus egg extracts. Genes Dev 2000;14:2745–56.[Abstract/Free Full Text]
  30. Kumagai A, Guo ZJ, Emami KH, et al. The Xenopus Chk1 protein kinase mediates a caffeine-sensitive pathway of checkpoint control in cell-free extracts. J Cell Biol 1998;142:1559–69.[Abstract/Free Full Text]
  31. Lukas J, Lukas C, Bartek J. Mammalian cell cycle checkpoints: signalling pathways and their organization in space and time. DNA Repair 2004;3:997–1007.[CrossRef][Medline]
  32. Nebreda AR, Porras A. p38 MAP kinases: beyond the stress response. Trends Biochem Sci 2000;25:257–60.[CrossRef][Medline]
  33. Raman M, Earnest S, Zhang K, et al. TAO kinases mediate activation of p38 in response to DNA damage. EMBO J 2007;26:2005–14.
  34. Mikhailov A, Shinohara M, Rieder CL. Topoisomerase II and histone deacetylase inhibitors delay the G2/M transition by triggering the p38 MAPK checkpoint pathway. J Cell Biol 2004;166:517–26.[Abstract/Free Full Text]
  35. Hwang A, Maity A, McKenna WG, et al. Cell cycle-dependent regulation of the cyclin B1 promoter. J Biol Chem 1995;270:28419–24.[Abstract/Free Full Text]
  36. Hwang A, McKenna WG, Muschel RJ. Cell cycle-dependent usage of transcriptional start sites. A novel mechanism for regulation of cyclin B1. J Biol Chem 1998;273:31505–9.[Abstract/Free Full Text]
  37. Wang W, Caldwell MC, Lin S, et al. HuR regulates cyclin A and cyclin B1 mRNA stability during cell proliferation. EMBO J 2002;19:2340–50.[CrossRef]
  38. Zhou BB, Bartek J. Targeting the checkpoint kinases: chemosensitization versus chemoprotection. Nat Rev Cancer 2004;4:216–25.[CrossRef][Medline]
  39. Shao J, Zhou B, Chu B, et al. Ribonucleotide reductase inhibitors and future drug design. Curr Cancer Drug Targets 2006;6:409–31.[CrossRef][Medline]



This article has been cited by other articles:


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
 JCBHome page
A. Lindqvist, V. Rodriguez-Bravo, and R. H. Medema
The decision to enter mitosis: feedback and redundancy in the mitotic entry network
J. Cell Biol., April 20, 2009; 185(2): 193 - 202.
[Abstract] [Full Text] [PDF]

Home page
 Clin. Cancer Res.Home page
S. Ashwell and S. Zabludoff
DNA Damage Detection and Repair Pathways--Recent Advances with Inhibitors of Checkpoint Kinases in Cancer Therapy
Clin. Cancer Res., July 1, 2008; 14(13): 4032 - 4037.
[Abstract] [Full Text] [PDF]

This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Supplementary Data
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