Molecular Cancer Therapeutics AM No Date
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 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 Yan, T.
Right arrow Articles by Kinsella, T. J.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Yan, T.
Right arrow Articles by Kinsella, T. J.
Mol Cancer Ther. 2004;3:1147-1157
© 2004 American Association for Cancer Research

CHK1 and CHK2 are differentially involved in mismatch repair–mediated 6-thioguanine-induced cell cycle checkpoint responses

Tao Yan, Anand B. Desai, James W. Jacobberger, R. Michael Sramkoski, Tamalette Loh and Timothy J. Kinsella

Department of Radiation Oncology, Case Comprehensive Cancer Center, University Hospitals of Cleveland and Case Western Reserve University School of Medicine, Cleveland, Ohio

Requests for reprints: Timothy J. Kinsella, Department of Radiation Oncology, LTR 6068, University Hospitals of Cleveland/Ireland Cancer Center, 11100 Euclid Avenue, Cleveland, OH 44106-6068. Phone: 216-844-2530; Fax: 216-844-4799. E-mail: timothy.kinsella{at}uhhs.com


   Abstract
Top
 Abstract
Introduction
 Experimental Procedures
 Results
 Discussion
 References
 
The DNA mismatch repair (MMR) system plays an important role in mediating a G2-M checkpoint arrest and subsequent cell death following treatment with a variety of chemotherapeutic agents. In this study, using 6-thioguanine (6-TG) as a mismatch-inducing drug, we examine the role of ataxia telangiectasia mutated (ATM)/CHK2 and ATM and Rad-3 related (ATR)/CHK1 signaling pathways in MMR-mediated cell cycle responses in MMR-proficient human colorectal cancer RKO cells. We show that, in response to 6-TG (3 µmol/L x 24 hours), activating phosphorylation of CHK1 at Ser317 [CHK1(pS317)] and CHK2 at Thr68 [CHK2(pT68)] are induced differentially during a prolonged course (up to 6 days) of MMR-mediated cell cycle arrests following 6-TG treatment, with CHK1(pS317) being induced within 1 day and CHK2(pT68) being induced later. Using chemical inhibitors and small interfering RNA of the signaling kinases, we show that a MMR-mediated 6-TG-induced G2 arrest is ATR/CHK1 dependent but ATM/CHK2 independent and that ATR/CHK1 signaling is responsible for both initiation and maintenance of the G2 arrest. However, CHK2(pT68) seems to be involved in a subsequent tetraploid G1 arrest, which blocks cells that escape from the G2-M checkpoint following 6-TG treatment. Furthermore, we show that CHK2 is hyperphosphorylated at later times following 6-TG treatment and the phosphorylation of CHK2 seems to be ATM independent but up-regulated when ATR or CHK1 is reduced. Thus, our data suggest that CHK1(pS317) is involved in a MMR-mediated 6-TG-induced G2 arrest, whereas CHK2(pT68) seems to be involved in a subsequent tetraploid G1-S checkpoint. The two signaling kinases seem to work cooperatively to ensure that 6-TG damaged cells arrest at these cell cycle checkpoints.


   Introduction
Top
 Abstract
 Introduction
Experimental Procedures
 Results
 Discussion
 References
 
The DNA mismatch repair (MMR) system is used by cells to protect genomic integrity from DNA replication errors (1–4). Loss of human MMR (particularly hMSH2 and hMLH1 deficiency) is associated with hereditary nonpolyposis colon cancer and is also found in many types of sporadic human cancers due to hypermethylation silencing (5–9). The functional units of hMMR are the heterodimers hMutS{alpha} (hMSH2-hMSH6), hMutSß (hMSH2-hMSH3), hMutLß (hMLH1-hPMS2), and hMutL{alpha} (hMLH1-hPMS1). The two hMutS heterodimers recognize and bind mismatches and insertion-deletion loops, whereas the two hMutL heterodimers recruit downstream repair complex proteins to the mismatch sites (2, 4). Although the primary function of hMMR is to correct inaccurately replicated DNA, it is also involved in cellular responses to a variety of chemotherapeutic drugs. The involvement of MMR in a drug response is usually not advantageous to cells because MMR-proficient (MMR+) cells have been shown to be sensitive to killing by several different classes of chemotherapeutic drugs including 6-thioguanine (6-TG), N-methyl-N'-nitro-N-nitrosoguanidine, methylnitrosourea, temozolomide, Adriamycin, procarbazine, busulfan, etoposide, cisplatin, carboplatin, benzopyrene, 5-fluorouracil, and 5-fluorodeoxyuridine (10–15). The increased cell death in MMR+ cells is usually preceded by an increased and prolonged G2-M arrest following drug exposure. In contrast, MMR-deficient (MMR–) cells show variable levels of in vitro/in vivo resistance to these chemotherapeutic drugs and show no effective G2-M arrest response. Our recent study, using 6-TG as a DNA mismatch-inducing drug, indicated that persistent DNA single-strand breaks produced during MMR processing of the 6-TG mismatches are likely initial chemical signals to a MMR-mediated G2-M arrest (16). No temporal correlation between DNA double-strand breaks and the 6-TG-induced G2-M arrest was found (16).

The protein serine-threonine kinases ataxia telangiectasia mutated (ATM) and ATM and Rad-3 related (ATR) have been known to play pivotal roles in human cell cycle checkpoint responses to DNA damage (17, 18). Whereas ATM has a well-acknowledged role in response to ionizing radiation–induced double-strand breaks, ATR seems to respond to a variety of DNA-damaging agents, including its established involvement in the cell cycle response to damage by UV light and alkylating agents as well as to DNA replication blockage (17, 18). ATM and ATR have several shared substrates and potentially their functions can overlap or act in a sequential manner (19–22). With respect to a G2-M checkpoint arrest, ATM and ATR have been shown to regulate it through phosphorylating and activating their downstream effector kinases CHK2 (by ATM) and CHK1 (by ATR), respectively, in response to genotoxic stress (18, 19). Activated CHK2 and CHK1 in turn phosphorylate and inactivate CDC25C phosphatase and hence maintain CDC2 in its phosphorylated and inactive form, which leads to a G2-M arrest (17, 18, 23).

Despite previously published data, it is not clearly understood how a MMR-mediated 6-TG-induced G2-M arrest is signaled. Indeed, many prior studies were carried out for only short (within 24 hours) time periods, although a MMR-mediated 6-TG-induced G2-M arrest can be sustained for several days as we recently reported (16). The main purpose of this study was to delineate the potential roles of ATM/CHK2 and ATR/CHK1 signaling pathways, with an emphasis on CHK2 and CHK1, in the MMR-mediated 6-TG-induced cell cycle checkpoint responses, in an attempt to identify molecular targets that can potentiate chemotherapeutic gain in MMR+ cells. We report a differential induction of activating phosphorylation of CHK1 at Ser317 [CHK1(pS317)] and CHK2 at Thr68 [CHK2(pT68)] in response to 6-TG treatment in MMR+ human colorectal cancer RKO cells. CHK1(pS317) increases early (significantly on day 1) following 6-TG treatment, which is temporally correlated with the onset of a 6-TG-induced G2 arrest. In contrast, CHK2(pT68) increases gradually at later times (days 2–6 following 6-TG treatment), which is temporally correlated with an increased tetraploid (4C) G1 arrest. Using chemical inhibitors and small interfering RNA (RNAi) knockdown of the signaling kinases, we show that a MMR-mediated 6-TG-induced G2 arrest is ATR/CHK1 dependent but ATM/CHK2 independent. However, an ATM-independent and ATR-independent phosphorylation of CHK2 at Thr68 seems to be involved in a subsequent 4C G1 arrest, which blocks cells that escape from the G2-M checkpoint following 6-TG treatment.


   Experimental Procedures
Top
 Abstract
 Introduction
 Experimental Procedures
Results
 Discussion
 References
 
Cell Culture
RKO is a human colorectal carcinoma cell line deficient in hMLH1 because of hypermethylation of the gene promoter region (24). The M4 cell line is a MMR+ RKO cell line derived by stable transfection of hMLH1 cDNA into the RKO cell line, and the MMR– V2 cell line is a negative control derived by transfection of an empty vector into the RKO cell line. Both M4 and V2 cell lines were recently established and characterized for 6-TG cytotoxicity by our group (16) as well as the type and time course of DNA damage using alkaline comet assay (for DNA single-strand breaks) and neutral pulse-field gel electrophoresis (for DNA double-strand breaks) (16). Both M4 and V2 cells have similar cell population doubling times ({cong}20–22 hours). All cells were grown in DMEM (Mediatech, Herndon, VA) supplemented with 10% fetal bovine serum (Hyclone, Logan, UT), 2 mmol/L glutamine, and 0.1 mmol/L nonessential amino acids (Life Technologies, Rockville, MD) in 10% CO2 at 37°C.

Schedule of Drug Treatment
The M4 and V2 cells were seeded at 3 x 106 per 10 cm dish in 10 mL complete medium and allowed to attach and grow for ~15 hours. 6-TG (3 µmol/L) was added into the medium (t = day –1). After 24 hours, 6-TG was removed (t = day 0) and fresh complete medium was added with or without caffeine (2 mmol/L, Sigma Chemical Co., St. Louis, MO) or UCN-01 (7-hydroxystaurosporine, 100 nmol/L, a kind gift from Dr. J. Sarkaria, Mayo Clinic, Rochester, MN). The cells were harvested daily for the next 6 days (t = days 1–6) for multiple assays. We chose this sequential treatment schedule for two reasons: (a) caffeine and UCN-01 showed moderate cell growth inhibition in our preliminary experiments, which could affect 6-TG incorporation into DNA if used concomitantly, and (b) there was no measurable cell cycle response during the 24-hour treatment period with 6-TG (see Results). Therefore, it was possible to measure the effect of these chemical inhibitors when caffeine or UCN-01 was added following 6-TG removal.

Transfection of RNAi
The M4 cells (6 x 105 per well) were seeded into a six-well tissue culture plate and were growing at 50% confluence the following day when the RNAi transfections were begun. The RNAi (Dharmacon Research, Lafayette, CO) used were SMART pool products, each pool consisting of four selected RNAi duplexes to target a specific gene. The products used were as follows: ATM RNAi (M-003201-00-05), ATR RNAi (M-003202-01-05), CHK1 RNAi (M-003255-01-05), CHK2 RNAi (M-003256-00-05), and a negative control (D-001206-13-05). The transfections of RNAi were carried out using LipofectAMINE 2000 (Life Technologies). For ATM or ATR RNAi transfection, 50 nmol/L (final concentration) RNAi and 15 µL LipofectAMINE 2000 were added into 2 mL medium for 24 hours before the cells were reseeded for 6-TG treatment. For CHK1 or CHK2 RNAi transfection, the same transfection protocol was carried out once before the cells were reseeded for 6-TG treatment and every 2 days following the 24-hour 6-TG treatment. We used these different RNAi transfection schedules because a one-time transfection of RNAi was able to continuously reduce ATM or ATR expression for up to 6 days in M4 cells, whereas we needed a repeated transfection of RNAi every 2 days to reduce CHK1 and CHK2 reexpression in these cells. Repeated transfection itself using our protocol was not toxic to cells as monitored by transfection of a negative control RNAi.

Western Blotting Analyses
Western blotting analyses were carried out as described previously (16). ATM and ATR were separated on 6% SDS-PAGE and transferred to a membrane at 40 V at 4°C overnight. The antibodies used were as follows: ATM (Novus Biologicals, Littleton, CO); ATR (Serotec, Oxford, United Kingdom); hMSH2 (Calbiochem, San Diego, CA); actin (Sigma Chemical); CDC2(pY15), CHK1(pS317), and CHK2(pT68) (Cell Signaling, Beverly, MA); CDC25C, cyclin B1, cyclin E, p53, p21, CHK1, CHK2, and secondary antibody IgG-horseradish peroxidase conjugates (Santa Cruz Biotechnology, Santa Cruz, CA).

Flow Cytometry Analysis
Determination of the cell cycle profile was described previously (16). For multiparameter flow cytometry, 1 x 106 fixed cells were incubated with different primary and secondary antibodies, which were labeled with fluorescent dyes before the DNA was stained with Hoechst 33342 (Molecular Probes, Eugene, OR). The antibodies used were as follows: cyclin E and cyclin B1/FITC (BD PharMingen, San Diego, CA); rabbit anti-phospho-histone H3 (Upstate Biotechnology, Lake Placid, NY); goat anti-rabbit-R-PE (Caltag Laboratories, Burlingame, CA); and goat anti-rabbit Alexa 647 (Molecular Probes). The measurements were made on a BD LSR Flow Cytometer (Becton Dickinson, San Jose, CA) with excitation wavelengths of 488 nm (FITC and PE), 633 nm (Alexa 647), and 325 nm (Hoechst). The multiparameter flow cytometric data were analyzed with Winlist 5.0 software (Verity Software, Topsham, ME).

Statistics
The data, where applicable, represent means ± SE. Data were analyzed using the Student's t test. Bar graphs of flow cytometry data were usually derived from representative experiments and therefore are presented without error bars. The reason for this is that, although we observed the same trends in changes repeatedly, the fraction of cells arrested in G2-M varied from one experiments to another presumably because asynchronous cells were used and hence the amount of 6-TG incorporated into DNA varied.


   Results
Top
 Abstract
 Introduction
 Experimental Procedures
 Results
Discussion
 References
 
Caffeine and UCN-01 Abrogate a 6-TG-Induced G2-M Arrest in MMR+ RKO Cells and Accumulate Cells in the G1-S Checkpoint with Tetraploidy Status
The effect of chemical inhibitors of ATM and ATR signaling pathways on a 6-TG-induced G2-M arrest was determined in our isogenic RKO cell lines V2 and M4. As expected, 6-TG treatment induced a delayed yet significantly sustained tetraploid population [this undiscriminated tetraploid DNA population will be called 4C (25) in this study] in MMR+ M4 cells but not in MMR– V2 cells (Fig. 1A), consistent with our previous report that 6-TG was significantly more cytotoxic to the MMR+ M4 cells (16). Using our treatment regimen, an increased 4C population in the M4 cells started on day 1 following 6-TG removal, peaked on day 3, and gradually decreased thereafter presumably due to repopulation from a small fraction of surviving cells at the later times. Caffeine (2 mmol/L), an inhibitor of ATM and ATR, reduced the 4C peak in the M4 cells within the first 24 hours (Fig. 1A, day 1, 19% compared with 35% in 6-TG treated alone) following 6-TG removal but was associated with an enhanced 4C population at the later times (Fig. 1A, days 4–6, 72% on day 6 compared with 45% in 6-TG treated alone). The dynamic changes in the % 4C fraction in Fig. 1A following treatment with 6-TG alone or sequential treatment with 6-TG/caffeine in M4 cells is quantitated in Fig. 1B. UCN-01 (100 nmol/L), an inhibitor of CHK1, added immediately following the 24 hours 6-TG treatment showed a similar effect as caffeine (Fig. 1C and D). Both caffeine and UCN-01 potentiated 6-TG cytotoxicity in MMR+ M4 cells as expected (data not shown). However, caffeine (2 mmol/L) or UCN-01 (100 nmol/L) alone did not induce an enhanced 4C population nor cell death in the M4 or V2 cells. Furthermore, in contrast to M4 cells, which showed growth inhibition with most cells having 4C DNA in response to the treatment of 6-TG alone or 6-TG/caffeine, V2 cells continued to rapidly grow into confluence with most cells being in the 2C G1 phase when exposed to the same conditions (Fig. 1A).



View larger version (27K):
[in this window]
[in a new window]
[Download PPT slide]
  		Figure 1. Sequential treatment with 6-TG/caffeine or 6-TG/UCN-01 initially abrogates a 6-TG-induced 4C fraction in MMR+ RKO cells and then enhances the 4C population. A, cell cycle histograms of MMR-proficient (M4) and MMR-deficient (V2) cells following 6-TG alone (3 µmol/L x 24 hours) or sequential 6-TG/caffeine (CAF) treatment. 6-TG was added (day -1) into the medium ~15 hours after cell seeding and was replaced with fresh medium with or without caffeine (2 mmol/L) 24 hours later (day 0, schedule A). Controls were log-phase M4 or V2 cells without treatment. B, % Cells with 4C DNA. Summary of 4C fraction in total cells in A. C, cell cycle histograms of the M4 cells with sequential 6-TG/UCN-01 treatment (3 µmol/L x 24 hours per 100 nmol/L). D, % M4 cells with 4C DNA. Derived from the cell cycle profile in C. Cell cycle data are representative of experiments done at least three times.

 
We next focused on MMR+ M4 cells to explore MMR-mediated cell cycle checkpoint signaling in response to 6-TG alone versus sequential treatment using 6-TG/caffeine or 6-TG/UCN-01. To verify the cell cycle phases of the caffeine-enhanced or UCN-01-enhanced 4C population, we examined various cell cycle protein markers. As a key mediator of the G2-M transition, high levels of CDC2 phosphorylation at Tyr15 [CDC2(pY15)] and cyclin B1 are indicative of a G2-M checkpoint arrest. As shown in Fig. 2A, 6-TG alone (3 µmol/L x 24 hours) induced increased levels of CDC2(pY15) and cyclin B1, which were sustained throughout the period of 6 days following 6-TG treatment. The increased CDC2(pY15)/cyclin B1 (Fig. 2A) was temporally correlated with an increased 4C population in M4 cells assayed with flow cytometry (Fig. 1A and B), confirming that 6-TG induced a sustained G2-M checkpoint arrest in the MMR+ M4 cells. In contrast, with sequential treatment of 6-TG and caffeine (2 mmol/L caffeine was added on day 0 immediately following removal of 6-TG and remained in the medium for the entire 6 days), the levels of CDC2(pY15)/cyclin B1 were only found to transiently increase on day 2 and then diminished dramatically (Fig. 2A), suggesting that the 4C population enhanced by caffeine did not remain at the G2-M checkpoint. However, the levels of cyclin E, which is known to accumulate at the G1-S border, were significantly higher in cells treated with 6-TG/caffeine (Fig. 2A), suggesting that the sequential treatment of 6-TG/caffeine may arrest M4 cells at the G1-S border with tetraploidy status, although most cells were mononucleate as visualized under a fluorescent microscope (data not shown). We also noted that p53 accumulated in M4 cells in response to 6-TG treatment regardless of the presence or absence of caffeine (Fig. 2A). Notably, all four cell cycle proteins assayed did not change significantly during the 24-hour treatment with 6-TG (Fig. 2A, day 0). Sequential treatment of 6-TG/UCN-01 led to similar changes in the cell cycle marker proteins in M4 cells (Fig. 2B), as found with sequential 6-TG/caffeine treatment (Fig. 2A).



View larger version (38K):
[in this window]
[in a new window]
[Download PPT slide]
  		Figure 2. 6-TG treatment arrests M4 cells in G2, whereas sequential treatment with 6-TG/caffeine or 6-TG/UCN-01 leads to accumulation of 4C G1 cells. A, western blots of cell cycle marker proteins in the cells treated with 6-TG (3 µmol/L x 24 hours) alone or with sequential 6-TG/caffeine similar to Fig. 1A. NT, log-phase M4 cells without treatment. B, cell cycle marker proteins in the M4 cells with sequential 6-TG/UCN-01 treatment similar to Fig. 1C. C, flow cytometric data show MPM2+ cells in total cells with or without nocodazole trapping. Cells were treated with 6-TG alone or 6-TG/caffeine as mentioned in Fig. 1A. Nocodazole was added to cells 16 hours prior to each cell harvest. Forty-percent control cells are MPM2+ with nocodazole trapping. Data are representative of two individual experiments.

 
To determine dynamic changes in cell cycle progress through the G2-M checkpoint on treatment of 6-TG/caffeine, we used nocodazole trapping and mitotic marker MPM2 staining of mitotic cells. The cells were harvested daily following 6-TG removal with or without caffeine present. Nocodazole was added 16 hours prior to each cell harvest. A representative result is presented in Fig. 2C. Consistent with the Western blotting data (Fig. 2A, day 0), cell cycle progress largely remained and cells vigorously entered M phase during the 24-hour treatment with 6-TG (Fig. 2C, day 0). Cell cycle progress was significantly reduced on day 1 after 6-TG removal and remained low through day 4 (Fig. 2C), consistent with persistence of a G2 arrest (Fig. 1A and B). In the presence of caffeine, the cells continued to enter the M phase on day 1 following 6-TG removal, though with a reduced rate, confirming abrogation of the G2-M checkpoint. However, on days 2 to 4, although 6-TG/caffeine resulted in more MPM2+ cells than 6-TG treatment alone at any given time (without nocodazole), nocodazole trapping only slightly increased MPM2+ cells. This observation suggests that either the cell cycle progress slowed down dramatically or the mechanism of MPM2+ registration was altered in these endoreplicating cells.

Together, these results suggest that ATM/CHK2 and/or ATR/CHK1 may be activated in the signaling of a MMR-mediated 6-TG-induced G2 arrest and that the M4 cells, which escape the G2-M checkpoint and fail to undergo normal mitosis following 6-TG/caffeine or 6-TG/UCN-01 treatment, subsequently arrest in G1 with a 4C DNA content.

Multiparameter Flow Cytometry Reveals That Sequential Treatment with 6-TG/Caffeine or 6-TG/UCN-01 Results in a Similar Extent of Tetraploid G1 Cells in MMR+ RKO Cells
To confirm and quantify 4C G1 cells in the total 4C cell population following the sequential drug treatments in the M4 cells, we carried out a multiparameter flow cytometric assay to measure DNA content, cyclin B1, cyclin A, and phospho-S10-histone H3 [known as a mitosis marker (26)] simultaneously. The M4 cell samples were harvested on day 4 following a 24-hour 6-TG treatment, when CDC2(pY15)/cyclin B1 had dramatically decreased, whereas a 4C cell population had increased in the presence of caffeine or UCN-01 (Figs. 1 and 2A and B). Figure 3A shows a histogram of cyclin B1 versus DNA, which distinguishes G2 cells that express a high level of cyclin B1 from 4C G1 cells that are essentially negative for cyclin B1 expression. Figure 3A also shows a histogram of phospho-S10-histone H3 versus DNA, which distinguishes highly expressing mitotic 4C cells. Additionally, the bottom panel of Fig. 3A illustrates a histogram of DNA versus cyclin A that is high in S, G2, and prophase cells. Cyclin B and Cyclin A serves as a double criteria to verify 4C G1 cells that are counted as those which have both low cyclin A and low cyclin B. These multiparameter flow data from Fig. 3A are summarized in Fig. 3B. At this time point, the % mitotic cells were extremely low in all treated M4 cells compared with the control cells, confirming that the drug treatments did not result in cells accumulating in M phase. These data also show that 6-TG alone results in some degree of 4C G1 cells (Fig. 3B, 38% 4C G1 versus 62% G2 of the total 4C population) on day 4 following 6-TG removal, whereas sequential treatment with 6-TG/caffeine or 6-TG/UCN-01 leads to a significantly increased tetraploid G1 population in total 4C cells on day 4 (Fig. 3B, 81% 4C G1 cells versus 19% G2 cells with 6-TG/caffeine treatment and 87% 4C G1 cells versus 13% G2 cells with 6-TG/UCN-01 treatment). Notably, chemical CHK1 inhibition (by UCN-01) alone results in a similar fraction of a 4C G1 population compared with chemical inhibition of both ATM and ATR (by caffeine).



View larger version (41K):
[in this window]
[in a new window]
[Download PPT slide]
  		Figure 3. Multiparameter flow cytometric assay reveals that sequential treatment with 6-TG/caffeine or 6-TG/UCN-01 in M4 cells results in a similar 4C G1 population. A, cell cycle histograms show a marked increase in a 4C G1 population on day 4 following 6-TG removal with sequential treatment using 6-TG/caffeine (CAF) or 6-TG/UCN-01 (UCN). B, flow cytometry data from Fig. 3A. Data are representative of three individual experiments.

 
The MMR-Mediated 6-TG-Induced G2 Arrest Signaling Mechanism Is Long-lived and Its Inhibition by Caffeine and UCN-01 Is Reversible
We next questioned whether the G2-M checkpoint signaling mechanism was responsible for both initiation and maintenance of a MMR-mediated 6-TG-induced G2 arrest. To address this question, the MMR+ M4 cells were treated with 6-TG for 24 hours, and caffeine or UCN-01 was added to the medium at different times following 6-TG removal including immediately [day 0, this is a schedule (schedule A) used throughout our study], on day 1 (schedule B), or on day 2 (schedule C). Caffeine or UCN-01 remained in the medium until the M4 cells were harvested and analyzed. These drug treatment schedules are summarized in the top panel of Fig. 4. Using Western blotting analyses, a similar reduction of CDC2(pY15) was observed with all three schedules (Fig. 4A and B, see schedule A results in Fig. 2A and B), indicating that the G2 arrest in M4 cells was abrogated by caffeine and UCN-01 and that response of the signaling mechanism to these chemical inhibitors could be initiated over a long (at least 24–48 hours) period following 6-TG exposure. However, when caffeine or UCN-01 was added immediately following 6-TG treatment (day 0) and removed on day 2 (schedule D), the CDC2(pY15) levels remained elevated to day 6 (Fig. 4A and B), similar to 6-TG treatment alone (Fig. 2A), indicating that a partial G2 arrest persisted in these MMR+ cells in spite of caffeine or UCN-01 treatment for the first 2 days following 6-TG removal. The cell cycle assay using cyclin B1 staining for G2 cells (Fig. 4C) showed that, whereas the continued presence of UCN-01 (schedules B and C) significantly reduced 6-TG-induced G2 cells to <10% of the total 4C cells (day 6), a significant portion of the G2 cells were sustained after UCN-01 was removed (schedule D). Thus, the Western blotting data (Fig. 4B) and the cell cycle data (Fig. 4C) are mutually supportive and suggest that the signaling mechanism that triggers the MMR-mediated 6-TG-induced G2 arrest is also responsible for the maintenance of the prolonged G2 arrest. These data further indicate that caffeine or UCN-01 inhibition of the MMR-mediated 6-TG-induced G2-M checkpoint arrest is reversible once the inhibitors are removed, at least within the time frame of 48 hours after 6-TG removal. These data are consistent with the report by Sarkaria et al. (27) that the inhibitory effects of caffeine on ATM and ATR are reversible.



View larger version (30K):
[in this window]
[in a new window]
[Download PPT slide]
  		Figure 4. The 6-TG-induced signaling in MMR+ RKO cells is long-lived and its inhibition by caffeine or UCN-01 is reversible. A, western blots of CDC2(pY15). Caffeine was added on day 1 (schedule B) or day 2 (schedule C) following 6-TG removal and remained in the medium, or caffeine was added on day 0 and removed on day 2 (schedule D). The M4 cells were harvested daily. See Fig. 2A for 6-TG treatment alone and schedule A of 6-TG/caffeine. B, western blots of CDC2(pY15). UCN-01 was added on day 1 (schedule B) or day 2 (schedule C) following 6-TG removal and remained in the medium, or UCN-01 was added on day 0 and removed on day 2 (schedule D). The M4 cells were harvested daily. See Fig. 2B for schedule A of 6-TG/UCN-01. C, % G2 cells (as cyclin B1 positive) in the total 4C population of M4 cells assayed with dual-flow cytometry following the different 6-TG/UCN-01 treatment schedules. Data are representative of two individual experiments.

 
CHK1(pS317) and CHK2(pT68) Are Differentially Regulated in MMR+ RKO Cells following 6-TG, 6-TG/Caffeine, or 6-TG/UCN-01 Treatment
To distinguish the role of ATM and/or ATR pathways in a MMR-mediated 6-TG-induced G2 arrest, we examined the downstream effectors of ATM and ATR, CHK2(pT68) and CHK1(pS317), respectively, after treatment of MMR+ M4 cells with 6-TG alone or sequential treatment with 6-TG/caffeine or 6-TG/UCN-01 for up to 4 days. As shown in Fig. 5, CHK1(pS317) was significantly increased on day 1 following 6-TG removal paralleling the onset of the G2-M checkpoint arrest and was decreased in the following days. In contrast, CHK2(pT68) was not detectable until day 2 following 6-TG removal and was maximal on day 4. The time course of CHK2(pT68) induction did not correlate with the time course of the G2 arrest in the MMR+ M4 cells. With 6-TG/caffeine treatment, CHK1(pS317) was moderately increased on day 1 but diminished on days 3 and 4. With sequential 6-TG/UCN-01 treatment, CHK1(pS317) was detectable although the activity was presumably inhibited by UCN-01. Surprisingly, a significant increase in CHK2(pT68) levels was found on day 2 under the continuous presence of caffeine or UCN-01, with strikingly high levels found on day 4 when most M4 cells were in 4C G1. Particularly, hyperphosphorylation of CHK2 at Thr68 was found on days 3 and 4 with 6-TG/UCN-01 treatment. These data suggest that the 6-TG-induced G2 arrest in MMR+ RKO cells is temporally correlated with an increased CHK1(pS317) level, whereas the 4C G1 arrest is temporally correlated with an increased CHK2(pT68) level.



View larger version (51K):
[in this window]
[in a new window]
[Download PPT slide]
  		Figure 5. Western blots show that CHK1(pS317) and CHK2(pT68) are differentially induced following 6-TG, 6-TG/caffeine (CAF), or 6-TG/UCN-01 treatments in MMR+ RKO cells as described in Fig. 1. NT, log-phase M4 cells without treatment. Data are representative of two individual experiments.

 
RNAi Knockdown of the Individual Signaling Kinases in MMR+ RKO Cells Shows That the MMR-Mediated 6-TG-Induced G2 Arrest Is ATR/CHK1 Dependent but ATM/CHK2 Independent
Because caffeine and UCN-01 have been reported to inhibit both ATR and ATM signaling pathways (27, 28), we used RNAi to further verify the roles of ATR/CHK1 and ATM/CHK2 in the MMR-mediated 6-TG-induced G2 arrest in MMR+ M4 cells. The effect of transfection of the different RNAi on CHK1(pS317) and CHK2(pT68) in the absence of 6-TG was examined. At 24 hours after a 24-hour RNAi transfection, RNAi knockdown of ATM and CHK2 enhanced CHK1(pS317), whereas RNAi knockdown of CHK1 enhanced CHK2(pT68) (Fig. 6A). However, these changes are minor and do not account for the changes in response to the drug treatments. We examined the combined treatment of RNAi and 6-TG. As shown in Fig. 6B, ATM protein was reduced to a level that was barely detectable and ATR protein was reduced to <10% initially and to <25% on day 4. When examining cell cycle proteins compared with those treated with a negative control RNAi, we found following differential responses to 6-TG in the ATR or ATM knockdown M4 cells (Fig. 6B). First, decreased CDC2(pY15) and increased cyclin E were found with an ATR knockdown but not with an ATM knockdown. Second, hyperphosphorylation of CHK2(pT68) was found with an ATR knockdown but not with an ATM knockdown. Third, decreased CHK1(pS317) was found with an ATR knockdown, whereas hyperphosphorylation of CHK1(pS317) was found on day 1 after 6-TG removal with an ATM knockdown. Fourth, p53 increased under all treatment conditions and p21, a downstream cell cycle effector of p53, increased concomitantly. Finally, the ATM protein level decreased on day 1 following 6-TG removal (Fig. 6B) when the G2-M checkpoint was activated. Consistent with the Western blotting data, the dual-parameter flow cytometric assay showed that an ATM knockdown in M4 cells did not change the extent of a 6-TG-induced G2 (cyclin B1 staining) arrest, whereas an ATR knockdown in M4 cells significantly decreased the % G2 cells in total 4C cells on days 2 to 4 after 6-TG removal (Fig. 6C).



View larger version (38K):
[in this window]
[in a new window]
[Download PPT slide]
  		Figure 6. RNAi knockdown of ATM or ATR in MMR+ RKO cells shows that a MMR-mediated 6-TG-induced G2 arrest correlates with ATR but not ATM. A, western blots show the effect of transfection of individual RNAi on CHK1(pS317) and CHK2(pT68) in the absence of 6-TG. B, western blots of G2-M cell cycle checkpoint signaling-related proteins show the changes following ATR knockdown but not following ATM knockdown compared with a negative control RNAi. MSH2, loading control for ATM and ATR that was separated on a 6% SDS-PAGE. NT, log-phase M4 cells without treatment. Data are representative of two individual experiments. C, % G2 cells in the total 4C population of M4 cells identified by dual-flow cytometry with cyclin B1 and propidium iodide double staining.

 
Figure 7A shows Western blotting of related cell cycle proteins in response to 6-TG treatment in M4 cells when CHK1 protein levels were reduced to <25%. When compared with the negative control RNAi, a CHK1 knockdown in M4 cells led to hyperphosphorylated CHK2(pT68), decreased CDC2(pY15)/cyclin B1, and increased cyclin E. However, p53 increased similarly in both control and CHK1 knockdown M4 cells. Figure 7B shows that the M4 cells with a CHK2 protein knockdown to <25% did not change the response pattern of the cell cycle proteins to 6-TG compared with those transfected with the negative control RNAi. The flow cytometric assays showed that a CHK2 knockdown in M4 cells did not change the cell cycle response profiles, whereas a CHK1 knockdown significantly decreased the % G2 cells in the total 4C cell population (Fig. 7C), a pattern found in caffeine and UCN-01 treatments in the M4 cells as well as in the ATR knockdown.



View larger version (38K):
[in this window]
[in a new window]
[Download PPT slide]
  		Figure 7. RNAi knockdown of CHK1 or CHK2 in MMR+ RKO cells shows that a MMR-mediated 6-TG-induced G2 arrest correlates with CHK1 but not CHK2. A,western blots show the changes in cell cycle proteins in 6-TG-treated CHK1 knockdown cells compared with the response in a control. NT, log-phase M4 cells without treatment. B, western blots show the CHK2 knockdown did not result in changes in cell cycle proteins in 6-TG-treated cells. Data are representative of two individual experiments. C, % G2 cells in the total 4C population of M4 cells identified by dual-flow cytometry with cyclin B1 and propidium iodide double staining.

 
RNAi Knockdown of ATR or CHK1 but Not ATM or CHK2 Enhances 6-TG Cytotoxicity in MMR+ RKO Cells
We reported previously that a MMR-mediated 6-TG-induced G2 arrest leads to significant cell growth inhibition and eventually cell death in the MMR+ M4 cells (16). In this study, we found that both caffeine and UCN-01 potentiated 6-TG cytotoxicity in MMR+ M4 cells (data not shown). We also assessed cytotoxicity to 6-TG in M4 cells following RNAi knockdown of ATR, ATM, CHK1, or CHK2. When compared with M4 cells treated with the negative control RNAi and 6-TG, which, like 6-TG treatment alone, show growth inhibition as reported previously (16), a knockdown of ATR/CHK1 protein in M4 cells results in an increased cell death following 6-TG treatment, whereas a knockdown of ATM/CHK2 protein in the M4 cells does not enhance cell death after 6-TG treatment (Fig. 8).



View larger version (37K):
[in this window]
[in a new window]
[Download PPT slide]
  		Figure 8. RNAi knockdown of ATR or CHK1 but not ATM or CHK2 enhances 6-TG cytotoxicity in MMR+ M4 cells. M4 cells transfected with specific RNAi and treated with 6-TG was compared with the control, which are M4 cells transfected with a nonspecific RNAi and treated with 6-TG (3 µmol/L x 24 hours). Cell number at each time point expressed as a percentage of the 6-TG-treated control. *, P < 0.1, significant differences.

 

   Discussion
Top
 Abstract
 Introduction
 Experimental Procedures
 Results
 Discussion
References
 
In this study, we show that CHK1 and CHK2 are differentially involved in MMR-mediated cell cycle checkpoint responses to 6-TG damage. We show that chemical inhibition or knockdown of ATR or CHK1 in the MMR+ RKO cells leads to a reduction in a MMR-mediated 6-TG-induced G2 arrest and an enhancement of a 4C G1 population, whereas chemical inhibition or knockdown of ATM or CHK2 does not result in similar effects. These data indicate that a MMR-mediated 6-TG-induced G2-M checkpoint arrest is ATR/CHK1 dependent but ATM/CHK2 independent. However, CHK2 was phosphorylated at Thr68 in response to 6-TG when ATM was inhibited or knocked down in MMR+ RKO cells and the CHK2(pT68) levels were positively correlated with the percentage of 4C G1 cells in the total 4C population, suggesting the involvement of an ATM-independent activation of CHK2 in a tetraploid G1-S checkpoint arrest following 6-TG treatment in MMR+ RKO cells.

MMR+ cells are highly sensitive to 6-TG, showing an initially prolonged G2-M arrest followed by cell death (16). 6-TG is a purine antimetabolite that has been in clinical use principally as an antileukemic agent for many years. 6-TG is incorporated into DNA by mammalian cells in place of dGTP during replication and methylated in situ to 6-thiomethylguanine by endogenous thiopurine S-methyltransferase. During the next replication cycle, 6-thiomethylguanine can pair with either a cytosine or a thymine base. Both 6-thiomethylguanine-cytosine and 6-thiomethylguanine-thymine mispairs are recognized by MMR (29–31). However, little is known about how the MMR-mediated 6-TG-induced G2-M checkpoint arrest is signaled. According to the futile cycle repair hypothesis (32) and our previous data (16), MMR attempts to process 6-TG mismatches in the daughter strands without removing damage from the parental strands, which leads to repetitive DNA single-strand breaks that may be a signal to a G2-M arrest. In other words, we postulate that 6-TG mismatches are not themselves signals to cell cycle arrest. Instead, the signal for a G2-M cell cycle checkpoint is most likely DNA single-strand breaks that are produced by futile MMR processing of 6-TG mismatches, as we recently published (16). Accordingly, 6-TG mismatches are well tolerated by MMR– cells such as the RKO V2 cells used in this study, which undergo neither G2 arrest (Fig. 1A and B) nor cytotoxicity (16).

The best-characterized cell cycle checkpoint signaling is the ATM/CHK2 pathway in response to ionizing radiation–induced DNA double-strand breaks. However, ATR/CHK1-dependent signaling has been intensely investigated and now is increasingly recognized to play an important role in the G2-M checkpoint regulation in response to different forms of genotoxic stress (18, 33–43). ATR/CHK1 signaling seems to be triggered by single-strand DNA lesions that arise following UV irradiation, treatment with alkylating agents, or abortive DNA replication induced by aphidicolin. Additionally, the ATM/CHK2 and ATR/CHK1 signaling pathways have been reported to crosstalk or function cooperatively mainly through dual regulation of substrate phosphorylation by ATM and ATR in response to DNA damage by genotoxic agents (19–22). In the present study, we show that the ATR/CHK1 pathway is responsible for the signaling of the MMR-mediated 6-TG-induced G2 arrest. We show that, in the MMR+ M4 cells, 6-TG treatment results in an increased CHK1(pS317) level with a time course that parallels the time course of the 6-TG-induced activation of G2-M checkpoint. Additionally, we show that this increased CHK1(pS317) is diminished at later times after subsequent caffeine treatment when most M4 cells have escaped the G2-M checkpoint but are arrested in a tetraploid G1 phase. We further show that knockdown of ATR or CHK1 but not knockdown of ATM or CHK2 by RNAi abrogates the MMR-mediated 6-TG-induced G2 arrest in the MMR+ M4 cells. Finally, we show that the ATR/CHK1 signaling is rather long-lived, suggesting that the pathway is responsible not only for the initiation of a MMR-mediated 6-TG-induced G2 arrest but also for the maintenance of the G2 arrest. Our results showing that all four cell cycle proteins assayed did not change significantly during the 24-hour treatment with 6-TG (Fig. 2A, day 0) are in accordance with the proposed model that 6-TG incorporation itself is not a signal for a G2-M checkpoint arrest; instead, the signals are produced during MMR processing on the mismatches that are formed during the second cycle of DNA replication (16, 29).

In this study, we find a surprisingly strong induction of CHK2(pT68) in the MMR+ M4 cells at a later stage (≥2 days) following treatment with 6-TG, 6-TG/caffeine, or 6-TG/UCN-01, with the extent and timing paralleling the occurrence of a 4C G1 arrest (Figs. 3 and 5), suggesting an involvement of CHK2(pT68) in the tetraploid G1-S checkpoint. This induction seems to be, at least in part, ATM and ATR independent because it occurs in the presence of caffeine (Fig. 5). Its ATM independence was confirmed when ATM protein was undetectable with Western blotting after ATM RNAi transfection (Fig. 6B). Hyperphosphorylation of CHK2 at Thr68 is especially striking when CHK1 activity is inhibited by UCN-01 (Fig. 5) or by RNAi knockdown of ATR or CHK1 protein (Figs. 6B and 7A), suggesting a compensatory increased upstream kinase activity, be it either ATM or other redundant kinases. Although it is plausible to speculate an involvement of CHK2(pT68) in the tetraploid G1-S checkpoint, we did not observe a decrease in the percentage of 4C G1 accordingly when CHK2 protein was largely depleted (Fig. 7C). We assume that CHK2(pT68) is only one of the redundant factors that are involved in a 4C G1 arrest. The molecular mechanism of the ATM-independent CHK2 phosphorylation remains to be established.

Another interesting observation in this study is that the ATM protein level was initially decreased when measured immediately following the 24-hour 6-TG treatment (data not shown) as well as on day 1 after 6-TG removal in MMR+ M4 cells (Fig. 6B). The ATM protein increased back to the control level. It is of interest to know why ATM protein is down-regulated under this condition, because our current knowledge is that ATM is rapidly activated by autophosphorylation in response to DNA damage (44–46), while the ATM protein level is not a regulation mechanism.

Furthermore, the p53 protein level is found to be increased in MMR+ M4 cells under all treatment regimens in our study (Figs. 2A, 6B, and 7A and B), consistent with the notion that p53 is a shared substrate of ATM/CHK2 and ATR/CHK1. However, p53 was also up-regulated when both ATM and ATR were presumably inhibited following 6-TG/caffeine treatment, indicating that an ATM-independent and ATR-independent pathway may exist, which may be DNA-dependent protein kinase or the p38 kinase pathway. Our observation suggests that p53 responds as a general sensor to genotoxic stress but not as a specific factor in signaling of the MMR-mediated 6-TG-induced G2 arrest. Nevertheless, the concomitantly increased p21 (Fig. 6B) implies that p53/p21 may play a role in a subsequent 4C G1 arrest.

The enhancement of 6-TG cytotoxicity in MMR+ M4 cells with a knockdown of ATR or CHK1 (Fig. 8) in this study supports the futile cycle repair model and our previous data that DNA single-strand breaks produced by MMR processing of 6-TG mismatches are likely signals to G2-M checkpoint (16). The abrogation of this G2 arrest releases the damaged cells that are destined to undergo endoreplication. The eventual cell death following release from a MMR-mediated 6-TG-induced G2 arrest seems not to be a mitotic catastrophe because most 4C cells are G1 cells. The formation of 4C G1 may contribute to the enhanced cell death by 6-TG/caffeine or 6-TG/UCN-01. Theoretically, our data support a potential strategy of combining certain chemotherapeutic drugs and a G2-M checkpoint abrogator in MMR+ tumors to achieve a better therapeutic gain.

In summary, we show that, in response to MMR-mediated 6-TG genotoxicity, the DNA damage signaling kinases CHK1(pS317) and CHK(pT68) are differentially induced. CHK1(pS317) is involved in a MMR-mediated 6-TG-induced G2 arrest, whereas CHK2(pT68) seems to be involved in a subsequent 4C G1-S checkpoint, which arrests the cells that escape from the G2-M checkpoint. Our data suggest that the two kinases work cooperatively to ensure DNA damage-carrying cells are arrested in cell cycle checkpoints and the persistent arrest may lead to eventual cell death. Our additional observations that ATM protein levels are temporarily reduced at early times following 6-TG treatment, that CHK2(pT68) is induced in an ATM-independent manner, that CHK2 is hyperphosphorylated when ATR and CHK1 are down-regulated, and that p53 is increased under all treatment conditions in our study underscore the complex nature of signaling pathways involved in MMR processing of drug damage.


   Footnotes
 
Grant support: NIH grant CA84578 (T.J. Kinsella).

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 2/ 9/04; revised 6/ 8/04; accepted 6/18/04.


   References
Top
 Abstract
 Introduction
 Experimental Procedures
 Results
 Discussion
 References
 

  1. Kolodner RD, Marsischky GT. Eukaryotic DNA mismatch repair. Curr Opin Genet Dev 1999;9:89–96.[CrossRef][Medline]
  2. Modrich P, Lahue R. Mismatch repair in replication fidelity, genetic recombination, and cancer biology. Annu Rev Biochem 1996;65:101–33.[CrossRef][Medline]
  3. Umar A, Kunkel TA. DNA-replication fidelity, mismatch repair and genome instability in cancer cells. Eur J Biochem 1996;238:297–307.[Medline]
  4. Aquilina G, Bignami M. Mismatch repair in correction of replication errors and processing of DNA damage. J Cell Physiol 2001;187:145–54.[CrossRef][Medline]
  5. Peltomaki P. Role of DNA mismatch repair defects in the pathogenesis of human cancer. J Clin Oncol 2003;21:1174–9.[Abstract/Free Full Text]
  6. Jiricny J, Nystrom-Lahti M. Mismatch repair defects in cancer. Curr Opin Genet Dev 2000;10:157–61.[CrossRef][Medline]
  7. Yamamoto H, Sawai H, Weber TK, Rodriguez-Bigas MA, Perucho M. Somatic frameshift mutations in DNA mismatch repair and proapoptosis genes in hereditary nonpolyposis colorectal cancer. Cancer Res 1998;58:997–1003.[Abstract/Free Full Text]
  8. Salvesen HB, MacDonald N, Ryan A, et al. Methylation of hMLH1 in a population-based series of endometrial carcinomas. Clin Cancer Res 2000;6:3607–13.[Abstract/Free Full Text]
  9. Fleisher AS, Esteller M, Wang S, et al. Hypermethylation of the hMLH1 gene promoter in human gastric cancers with microsatellite instability. Cancer Res 1999;59:1090–5.[Abstract/Free Full Text]
  10. Fink D, Aebi S, Howell SB. The role of DNA mismatch repair in drug resistance. Clin Cancer Res 1998;4:1–6.[Abstract]
  11. Irving JA, Hall AG. Mismatch repair defects as a cause of resistance to cytotoxic drugs. Expert Rev Anticancer Ther 2001;1:149–58.[CrossRef][Medline]
  12. Li GM. The role of mismatch repair in DNA damage-induced apoptosis. Oncol Res 1999;11:393–400.[Medline]
  13. Tokunaga E, Oda S, Fukushima M, Maehara Y, Sugimachi K. Differential growth inhibition by 5-fluorouracil in human colorectal carcinoma cell lines. Eur J Cancer 2000;36:1998–2006.
  14. Meyers M, Wagner MW, Hwang HS, Kinsella TJ, Boothman DA. Role of the hMLH1 DNA mismatch repair protein in fluoropyrimidine-mediated cell death and cell cycle responses. Cancer Res 2001;61:5193–201.[Abstract/Free Full Text]
  15. Cejka P, Stojic L, Mojas N, et al. Methylation-induced G(2)/M arrest requires a full complement of the mismatch repair protein hMLH1. EMBO J 2003;22:2245–54.[CrossRef][Medline]
  16. Yan T, Berry SE, Desai AB, Kinsella TJ. DNA mismatch repair (MMR) mediates 6-thioguanine genotoxicity by introducing single-strand breaks to signal a G(2)-M arrest in MMR-proficient RKO cells. Clin Cancer Res 2003;9:2327–34.[Abstract/Free Full Text]
  17. Shiloh Y. ATM and related protein kinases: safeguarding genome integrity. Nat Rev Cancer 2003;3:155–68.[CrossRef][Medline]
  18. Abraham RT. Cell cycle checkpoint signaling through the ATM and ATR kinases. Genes Dev 2001;15:2177–96.[Free Full Text]
  19. 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]
  20. Tibbetts RS, Brumbaugh KM, Williams JM, et al. A role for ATR in the DNA damage-induced phosphorylation of p53. Genes Dev 1999;13:152–7.[Abstract/Free Full Text]
  21. Chen P, Gatei M, O'Connell MJ, et al. Chk1 complements the G2-M checkpoint defect and radiosensitivity of ataxia-telangiectasia cells. Oncogene 1999;18:249–56.[CrossRef][Medline]
  22. Hammond EM, Dorie MJ, Giaccia AJ. ATR/ATM targets are phosphorylated by ATR in response to hypoxia and ATM in response to reoxygenation. J Biol Chem 2003;278:12207–13.[Abstract/Free Full Text]
  23. Vermeulen K, Van Bockstaele DR, Berneman ZN. The cell cycle: a review of regulation, deregulation and therapeutic targets in cancer. Cell Prolif 2003;36:131–49.[CrossRef][Medline]
  24. Veigl ML, Kasturi L, Olechnowicz J, et al. Biallelic inactivation of hMLH1 by epigenetic gene silencing, a novel mechanism causing human MSI cancers. Proc Natl Acad Sci U S A 1998;95:8698–702.[Abstract/Free Full Text]
  25. Casenghi M, Mangiacasale R, Tuynder M, et al. p53-independent apoptosis and p53-dependent block of DNA rereplication following mitotic spindle inhibition in human cells. Exp Cell Res 1999;250:339–50.[CrossRef][Medline]
  26. Juan G, Traganos F, James WM, et al. Histone H3 phosphorylation and expression of cyclins A and B1 measured in individual cells during their progression through G2 and mitosis. Cytometry 1998;32:71–7.[CrossRef][Medline]
  27. Sarkaria JN, Busby EC, Tibbetts RS, et al. Inhibition of ATM and ATR kinase activities by the radiosensitizing agent, caffeine. Cancer Res 1999;59:4375–82.[Abstract/Free Full Text]
  28. Yu Q, La Rose J, Zhang H, Takemura H, Kohn KW, Pommier Y. UCN-01 inhibits p53 up-regulation and abrogates {gamma}-radiation-induced G(2)-M checkpoint independently of p53 by targeting both of the checkpoint kinases, Chk2 and Chk1. Cancer Res 2002;62:5743–8.[Abstract/Free Full Text]
  29. Swann PF, Waters TR, Moulton DC, et al. Role of postreplicative DNA mismatch repair in the cytotoxic action of thioguanine. Science 1996;273:1109–11.[Abstract]
  30. Griffin S, Branch P, Xu YZ, Karran P. DNA mismatch binding and incision at modified guanine bases by extracts of mammalian cells: implications for tolerance to DNA methylation damage. Biochemistry 1994;33:4787–93.[CrossRef][Medline]
  31. Waters TR, Swann PF. Cytotoxic mechanism of 6-thioguanine: hMutS{alpha}, the human mismatch binding heterodimer, binds to DNA containing S6-methylthioguanine. Biochemistry 1997;36:2501–6.[CrossRef][Medline]
  32. Karran P, Bignami M. DNA damage tolerance, mismatch repair and genome instability. BioEssays 1994;16:833–9.[CrossRef][Medline]
  33. Hekmat-Nejad M, You Z, Yee MC, Newport JW, Cimprich KA. Xenopus ATR is a replication-dependent chromatin-binding protein required for the DNA replication checkpoint. Curr Biol 2000;10:1565–73.[CrossRef][Medline]
  34. Guo Z, Kumagai A, Wang SX, Dunphy WG. 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]
  35. 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]
  36. Cortez D, Guntuku S, Qin J, Elledge SJ. ATR and ATRIP: partners in checkpoint signaling. Science 2001;294:1713–6.[Abstract/Free Full Text]
  37. 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]
  38. Unsal-Kacmaz K, Makhov AM, Griffith JD, Sancar A. Preferential binding of ATR protein to UV-damaged DNA. Proc Natl Acad Sci U S A 2002;99:6673–8.[Abstract/Free Full Text]
  39. Hammond EM, Denko NC, Dorie MJ, Abraham RT, Giaccia AJ. Hypoxia links ATR and p53 through replication arrest. Mol Cell Biol 2002;22:1834–43.[Abstract/Free Full Text]
  40. Cliby WA, Lewis KA, Lilly KK, Kaufmann SH. S phase and G2 arrests induced by topoisomerase I poisons are dependent on ATR kinase function. J Biol Chem 2002;277:1599–606.[Abstract/Free Full Text]
  41. Wang X, Khadpe J, Hu B, Iliakis G, Wang Y. An overactivated ATR/CHK1 pathway is responsible for the prolonged G2 accumulation in irradiated AT cells. J Biol Chem 2003;278:30869–74.[Abstract/Free Full Text]
  42. 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]
  43. Banin S, Moyal L, Shieh S, et al. Enhanced phosphorylation of p53 by ATM in response to DNA damage. Science 1998;281:1674–7.[Abstract/Free Full Text]
  44. Canman CE, Lim DS, Cimprich KA, et al. Activation of the ATM kinase by ionizing radiation and phosphorylation of p53. Science 1998;281:1677–9.[Abstract/Free Full Text]
  45. Bakkenist CJ, Kastan MB. DNA damage activates ATM through intermolecular autophosphorylation and dimer dissociation. Nature 2003;421:499–506.[CrossRef][Medline]
  46. Kozlov S, Gueven N, Keating K, Ramsay J, Lavin MF. ATP activates ataxia-telangiectasia mutated (ATM) in vitro. Importance of autophosphorylation. J Biol Chem 2003;278:9309–17.[Abstract/Free Full Text]



This article has been cited by other articles:


Home page
 Cancer Res.Home page
D. P. Turner, S. Cortellino, J. E. Schupp, E. Caretti, T. Loh, T. J. Kinsella, and A. Bellacosa
The DNA N-Glycosylase MED1 Exhibits Preference for Halogenated Pyrimidines and Is Involved in the Cytotoxicity of 5-Iododeoxyuridine.
Cancer Res., August 1, 2006; 66(15): 7686 - 7693.
[Abstract] [Full Text] [PDF]

Home page
 Hum Mol GenetHome page
S. Ziv, O. Brenner, N. Amariglio, N. I. Smorodinsky, R. Galron, D. V. Carrion, W. Zhang, G. G. Sharma, R. K. Pandita, M. Agarwal, et al.
Impaired genomic stability and increased oxidative stress exacerbate different features of Ataxia-telangiectasia
Hum. Mol. Genet., October 1, 2005; 14(19): 2929 - 2943.
[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 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 Yan, T.
Right arrow Articles by Kinsella, T. J.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Yan, T.
Right arrow Articles by Kinsella, T. J.

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