Molecular Cancer Therapeutics Funding
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 Google Scholar
Google Scholar
Right arrow Articles by Tu, L. C.
Right arrow Articles by Beerman, T. A.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Tu, L. C.
Right arrow Articles by Beerman, T. A.
Mol Cancer Ther. 2005;4:1175-1185
© 2005 American Association for Cancer Research

Hedamycin, a DNA alkylator, induces {gamma}H2AX and chromosome aberrations: Involvement of phosphatidylinositol 3-kinase–related kinases and DNA replication fork movement

Lan Chun Tu1, Sei ichi Matsui2 and Terry A. Beerman1

Departments of 1 Pharmacology and Therapeutics and 2 Cancer Genetics, Roswell Park Cancer Institute, Buffalo, New York

Requests for reprints: Terry A. Beerman, Department of Pharmacology and Therapeutics, Roswell Park Cancer Institute, Elm and Carlton Streets, Buffalo, NY 14263. Phone: 716-845-3443; Fax: 716-845-1575. E-mail: terry.beerman{at}roswellpark.edu


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Genotoxic treatments, such as UV light, camptothecin, and adozelesin, stall DNA replication and subsequently generate DNA strand breaks. Typically, DNA breaks are reflected by an increase in ataxia and Rad-related kinase (ATR)–regulated phosphorylation of H2AX ({gamma}H2AX) and require replication fork movement. This study examined the potential of the monofunctional DNA alkylating agent hedamycin, a powerful inhibitor of DNA replication, to induce DNA strand breaks, phosphorylated H2AX ({gamma}H2AX) foci, and chromosome aberrations. Hedamycin treatment of HCT116 carcinoma cells resulted in a rapid induction of DNA strand breaks accompanied by increasing H2AX phosphorylation and focalization. Unlike many other treatments that also stall replication, such as UV, camptothecin, and adozelesin, {gamma}H2AX formation was not suppressed in ATR-compromised cells but actually increased. Similarly, hedamycin induction of {gamma}H2AX is not dependent on ataxia telangiectasia mutated or DNA-protein kinase, and pretreatment of cells with the phosphatidylinositol 3-kinase–related kinase inhibitor caffeine did not substantially reduce induction of H2AX phosphorylation by hedamycin. Furthermore, the DNA replication inhibitor aphidicolin only modestly depressed hedamycin-induced {gamma}H2AX formation, indicating that hedamycin-induced DNA double-strand breaks are not dependent on fork progression. In contrast, camptothecin- and adozelesin-induced {gamma}H2AX was strongly suppressed by aphidicolin. Moreover, after 24 hours following a short-term hedamycin treatment, cells displayed high levels of breaks in interphase nuclear DNA and misjoined chromosomes in metaphase cells. Finally, focalization of a tightly bound form of Ku80 was observed in interphase cells, consistent with the subsequent appearance of chromosomal aberrations via abnormal nonhomologous end joining. Overall, this study has revealed a disparate type of DNA damage response to stalled replication induced by a bulky DNA adduct inducer, hedamycin, that seems not to be highly dependent on ATR or DNA replication.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
Treatments that induce replication stalling, albeit by diverse mechanisms, such as UV (1), hydroxyurea (2), or camptothecin (3), typically activate an ataxia and Rad-related kinase (ATR) to induce a phosphatidylinositol 3-kinase–related kinase (PIKK)–regulated checkpoint response (4) and also induce DNA breaks (5). Generally, loss of ATR impedes the DNA damage response to DNA double-strand breaks (DSB) and can result in increased sensitivity to agents that arrest replication because cell division can occur in the presence of damaged DNA (6, 7). Ultimately, failure to suppress cell cycle progression in the presence of sustained DNA DSBs can generate chromosomal abnormalities associated with genomic instability (8).

Following induction of DSBs, H2AX, a variant of a core histone, is almost immediately phosphorylated by PIKKs on Ser139 at its COOH-terminal tail and rapidly localizes to sites of breaks (9, 10) and promotes recruitment of other DNA damage response proteins, such as Mre11, Rad51, and Nbs1 (11–13). Because {gamma}H2AX formation is observed within seconds of induction of DNA breaks and the level of phosphorylation and intensity of immunolabeling increase linearly with the amount of damage, it is now a widely accepted marker of DSBs (9, 10).

DNA lesions, whether induced by depletion of deoxynucleotide triphosphate pools or photoproducts, activate an intra-S-phase checkpoint response, resulting in the blockage of late-firing origins and stabilization of stalled replication forks (14). However, this intra-S-phase checkpoint is unable to stop fork progression of early-firing origins, which were triggered before checkpoint activation (15, 16). Consequently, collisions between early-firing replication forks and hydroxyurea or UV-induced DNA lesions can result in DSBs and {gamma}H2AX formation and its nuclear foci (17). Unlike ionizing radiation, H2AX phosphorylation is regulated prominently by ATR, consistent with its role in sensing stalled replication forks, such as those generated by UV type lesions (17, 18).

The cellular responses to DNA-targeted alkylating drugs that alter DNA structure and impede its replication while indirectly inducing DNA DSBs remain relatively unknown. Typically, ATR activation is also involved in response to various types of bulky DNA adducts that lead to arrested replication forks (19). However, growing evidences revealed that lesion recognition is not sufficient for ATR activation and that single-strand DNA, resulting from replication or DNA repair process, is required for recruiting checkpoint proteins to damaged DNA (7, 20). Furthermore, based on H2AX phosphorylation, many nonradiomimetic DNA-damaging agents that do not directly induce breaks induce DSBs stemming from encounters of replication forks with preexisting nicks, gaps, or adducts (5, 21). For example, camptothecin, which induces cleavable complexes of topoisomerase I and DNA, also induces DNA breaks resulting from collisions between advancing replication forks and these complexes (22, 23). Similarly, DNA adducts induced by the monofunctional DNA alkylator adozelesin are not detected by the checkpoint response machinery until collisions of replication forks with the DNA lesions result in DSBs (24). For both agents, H2AX phosphorylation is suppressed by aphidicolin, a DNA polymerase inhibitor that blocks replication fork movement (23, 24).

We recently delineated the DNA damage responses induced by a pluramycin antibiotic, hedamycin (25). Hedamycin, like adozelesin, is a highly cytotoxic monofunctional alkylator (the concentration of drug required to kill 90% of the cells in a clonogenic survival assay is 0.38 nmol/L), but it also significantly distorts the DNA helix by threading intercalation (26). Like other DNA-damaging agents, hedamycin rapidly and strongly blocks DNA replication (25). However, it is not known whether its DNA adducts and stalled replication forks are converted into DNA strand breaks. One would speculate that hedamycin-induced DNA damage responses would be similar to other agents that induce bulky adducts; yet unexpectedly, DNA damage responses of hedamycin exhibit several atypical characteristics. For example, it does not induce G2 arrest but primarily S-phase accumulation and G1 arrest (25). What is particularly striking is that, unlike adozelesin or camptothecin, the cytotoxicity and cell cycle effects of hedamycin are not affected by the loss of ATR (25).

This study revealed that, whereas hedamycin induces DNA strand breaks and {gamma}H2AX formation, ATR suppression does not diminish either. Moreover, blockage of replication fork movement significantly reduced neither DNA breaks nor {gamma}H2AX formation. Hedamycin-induced DNA breaks are accompanied by chromosome abnormalities likely resulting from nonhomologous end joining (NHEJ). Hedamycin seems to be a unique agent for exploring cellular DNA damage response mechanisms that are associated with replication stalling and induction of DNA breaks and chromosomal aberrations.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Cell Lines
The HCT116 human colon carcinoma cell line (a gift from Dr. B. Vogelstein, Johns Hopkins Oncology Center, Baltimore, MD) was grown in McCoy's medium supplemented with 10% fetal bovine serum. Human glioma cell lines M059K [wild-type and DNA-protein kinase (DNA-PK) +] and M059J (DNA-PK–) were cultured in DMEM/F-12K (1:1) supplemented with 10% fetal bovine serum and 2x nonessential amino acids. Human lymphoblast cell lines GM00536B [wild-type and ataxia telangiectasia mutated (ATM) +] and GM01526E (ATM–) were maintained in RPMI supplemented with 15% heat-inactivated fetal bovine serum. Both glioma and lymphoblast cell lines were purchased from Coriell Institute for Medical Research (Camden, NJ), and all cell lines were kept at 37°C in a 5% CO2 incubator.

Chemicals
Hedamycin was provided by the National Cancer Institute. Hedamycin stock solutions (10 mmol/L) were prepared in water, aliquoted, and stored at –20°C. Subsequent dilutions were made in growth medium. Adozelesin (supplied by Pfizer, Inc., New York, NY) was dissolved in dimethylacetamide (2 mg/mL) and further diluted in DMSO to a final concentration of 1 nmol/L. Camptothecin, aphidicolin, caffeine, colcemid, Hoechst 33258, and ethidium bromide were purchased from Sigma Co. (St. Louis, MO). Camptothecin was aliquoted and stored frozen at 10 mmol/L in DMSO and further diluted in growth medium to a final concentration of 1 µmol/L before use. Aphidicolin was stored frozen at 1 mg/mL in ethanol and further diluted in growth medium before use. Caffeine was dissolved in growth medium at a stock concentration of 80 mmol/L before use. UV was given with a Stratalinker (Stratagene, La Jolla, CA) after cells were washed once with PBS. Oligofectamine was purchased from Invitrogen (Carlsbad, CA). ATR small interfering RNA (siRNA; ref. 27) was purchased from Dharmacon Research, Inc. (Lafayette, CO).

Antibodies
Anti-{gamma}H2AX and anti–proliferating cell nuclear antigen (PCNA) mouse monoclonal antibodies were purchased from Upstate Biotechnology, Inc. (Altham, MA). Anti-ATR goat polyclonal antibody was purchased from Santa Cruz Biotechnology (Santa Cruz, CA). Anti-Ku80 monoclonal antibodies were purchased from Sigma. Texas red–conjugated goat anti-mouse IgG was purchased from Pierce (Rockford, IL).

Drug Treatments
Cells were treated with hedamycin for 4 hours and either harvested directly or incubated in drug-free medium for another 24 hours after drug removal before harvesting for further evaluation. In some experiments, cells were pretreated with 16 mmol/L caffeine for 30 minutes or 0.5 µmol/L aphidicolin for 5 minutes before addition of hedamycin. If not indicated, cells were harvested 1 hour after exposure to 10 J/m2 UV, 4 hours after exposure to 5 nmol/L hedamycin, or 3 hours after exposure to 1 µmol/L camptothecin.

Alkaline Comet Assay
Cells were harvested by trypsinization and resuspended in PBS (28). Cell suspensions (1 x 105 cells) were mixed with 0.5% LMP agarose and spread on 1% agarose-precoated slides. After 30-minute incubation at 4°C, cells were lysed for 1 hour in alkaline buffer [2.5 mol/L NaCl, 0.1 mol/L EDTA, 10 mmol/L Tris-HCl (pH 10), 1% sarcosyl, 10% DMSO, 1% Triton X-100 (pH 10)]. After 20-minute equilibration in electrophoresis buffer [0.3 mol/L NaOH, 1 mmol/L EDTA (pH 13)], cells were subjected to electrophoresis at 28 V for 25 minutes under alkaline conditions at 4°C. After electrophoresis, slides were neutralized with 0.4 mol/L Tris (pH 7.4), fixed with methanol and ethanol, and dried overnight. Cells were then stained with 1 µg/mL ethidium bromide and analyzed using a fluorescence microscope Olympus BX40 (Melville, NY) with a Spot-RT digital camera and software (Webster, NY). At least 50 cells were evaluated per slide. Visual scoring of comet images using fluorescence microscopy has been described.3 Each of 50 comets per gel is assigned to a category based on the extent of the comet tail and classified from 0 to 4 depending on the relative intensity of DNA fluorescence in the tail (0 = undamaged, no DNA in tail; 4 = >80% of the DNA in tail). The cell number (in each category) times each score (0-4) then divided by the total cell number is used as a measure of the frequency of DNA breaks.

siRNA Transfection
Oligofectamine was used for transfection of siRNAs into HCT116 cells according to the protocols provided by Dharmacon. Expression of ATR was monitored by Western blotting. Following suppression of ATR (48 hours after transfection with siRNA), cells were exposed to hedamycin or UV irradiation.

Immunoblotting
Cells were washed twice with PBS and lysed at 4°C in a lysis buffer [50 mmol/L HEPES (pH 7.4), 4 mmol/L EDTA, 2 mmol/L EGTA, 1% Triton X-100, 50 mmol/L phenylmethylsulfonyl fluoride, 20 µg/mL leupeptin, 100 mmol/L Na3VO4]. Lysates were centrifuged at 14,000 rpm for 20 minutes. The protein concentration in the lysates was determined using the Bio-Rad (Hercules, CA) protein assay kit. Cell lysates were subjected to SDS-PAGE electrophoresis and transferred to a polyvinylidene difluoride membrane (Hybond-P, Amersham Pharmacia Biotech, Piscataway, NJ). The membrane was blocked with 4% nonfat milk in TBS [25 mmol/L Tris (pH 7.4), 125 mmol/L NaCl] for 1 hour at room temperature followed by incubation with primary antibodies against {gamma}H2AX, ATR, or PCNA. The membranes were washed thrice with TBS and then incubated with horseradish peroxidase–conjugated anti-goat or anti-mouse IgG for 1 hour. The immunoblot was then washed five times with TBS and developed using an enhanced chemiluminescence Western lightning kit (Perkin-Elmer, Boston, MA). Experiments were repeated at least thrice.

Indirect Immunofluorescence
Cells were grown on a coverslip, which was precoated with fetal bovine serum for 30 minutes before cell seeding. After drug treatment, the cells were washed with PBS, fixed with 2% formaldehyde for 20 minutes at room temperature followed by permeabilization with acetone for 3 minutes at –20°C, and then washed with PBS. The primary antibodies anti-{gamma}H2AX (1:300) or anti-Ku80 (1:50,000) monoclonal antibody were diluted in PBS containing 1% bovine serum albumin, applied to the coverslip, and kept overnight at 4°C. Unbound primary antibodies were washed off with PBS, and Texas red–labeled (1:600) secondary antibody with 10 mmol/L Hoechst 33258 (1:500) was applied for another 1 hour at room temperature. After a series of washes, the stained cells were mounted on slides, viewed using appropriate filters, and photographed by fluorescence microscopy with a Spot-RT digital camera. For Ku80 foci detection, cells were extracted with a lysis buffer containing 50 mmol/L HEPES (pH 7.4), 150 mmol/L NaCl, 1 mmol/L EDTA, and 0.5% NP40 supplemented with 10 mmol/L NaF, 1 mmol/L Na3VO4, and 10 µg/mL leupeptin for 30 minutes at 4°C and washed once with PBS before fixation. Each treatment was duplicated and the experiment was repeated at least thrice.

Spectral Karyotyping Analysis
HCT116 cells were treated with hedamycin for 4 hours followed by removal of free drug and further cultured in drug-free medium for 24 hours. At 2 hours before harvest, colcemid was added to a final concentration of 50 ng/mL. Cells were harvested by trypsinization, treated with hypotonic KCl solution (75 mmol/L) for 7 minutes, and fixed with methanol/glacial acetic acid (3:1). Slides were prepared by standard techniques and chromosomes were stained with 4',6-diamidino-2-phenylindole and inverted images were processed using Applied Spectral Imaging's EasyFISH software (29). One hundred metaphases per treatment condition were evaluated. Because untreated normal cells often display one to two gaps and/or breaks (mostly chromatid type), cells with more than two gaps/breaks were considered as metaphases with aberrations.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Short-term Treatment with Hedamycin Induces H2AX Phosphorylation and Focalization
DSBs stemming from replication arrest, such as those generated by treatments that induce bulky DNA lesions, such as UV and camptothecin, are generally detected by their ability to trigger H2AX phosphorylation and focalization at DNA damage sites (17, 30–32). To test if hedamycin, which also induces bulky DNA adducts, induces DNA DSBs based on phosphorylation of H2AX ({gamma}H2AX), cell lysates from HCT116 cells treated with hedamycin for 4 hours were analyzed by Western blotting. Figure 1A shows that the phosphorylation level of H2AX was increased in a concentration-dependent manner. A minimum induction was detected even at subnanomolar concentration (0.1 nmol/L) and phosphorylation levels approach a plateau at concentrations above 2.5 nmol/L. Whether phosphorylation of H2AX was accompanied by {gamma}H2AX foci formation was examined by immunofluorescence under the same treatment condition. {gamma}H2AX was stained with {gamma}H2AX antibody and chromosomes were stained with Hoechst 33258 (Fig. 1B shows representative images). The intensity of {gamma}H2AX foci seemed consistent with its phosphorylation levels.



View larger version (68K):
[in this window]
[in a new window]
[Download PPT slide]
  		Figure 1. Hedamycin-induced DNA strand breaks lead to {gamma}H2AX formation. HCT116 cells were treated with hedamycin for 4 h, after which cells were collected. A, Western blot showing a concentration-dependent increase of {gamma}H2AX. Cell lysates were prepared and analyzed by SDS-PAGE and Western blotting. Blots were probed with antibodies against {gamma}H2AX and PCNA. B, immunofluorescence showing focalization of {gamma}H2AX. Cells were fixed and immunostained with anti-{gamma}H2AX and anti-mouse IgG antibody conjugated with Texas red. Nuclei were stained with Hoechst 33258.

 
Repression of ATR Levels Does Not Decrease but Increase Hedamycin-Induced Phosphorylation of H2AX
The phosphorylation of H2AX in response to replication arrest-mediated DNA DSBs is dependent on the PIKK, ATR (33). To test the ATR dependency for hedamycin-induced phosphorylation of H2AX, siRNA interference was used to reduce ATR levels in HCT116 cells. Based on Western blotting, decreased levels of ATR were observed at 48 hours after transfection of a siRNA against ATR (Fig. 2A). Although H2AX phosphorylation levels increased albeit to a small degree in untreated siRNA-transfected cells, hedamycin-induced phosphorylation of H2AX was substantially increased compared with mock-transfected cells (Fig. 2A). Another cellular protein PCNA, involved in DNA replication, was unaffected by either drug treatment or siRNA transfection. The unanticipated increase in hedamycin-induced H2AX phosphorylation in the ATR-reduced cells seemed unrelated to the method of achieving ATR reduction or to the nature of HCT116 cells, because with UV, as shown by others, H2AX phosphorylation was greatly reduced in the absence of ATR compared with mock-transfected cells (Fig. 2B).



View larger version (34K):
[in this window]
[in a new window]
[Download PPT slide]
  		Figure 2. Reduction of ATR enhances H2AX phosphorylation in response to hedamycin. HCT116 cells were transfected with buffer (Mock) or siRNA against ATR (siATR) and continuously incubated with buffer or the siRNA for 48 h. At 48 h after transfection, cells were exposed to (A) hedamycin (HD) for 4 h or (B) UV irradiation (10 J/m2) and incubated for another 1 h. Cell lysates were prepared and proteins were separated by SDS-PAGE and immunoblotted with anti-{gamma}H2AX, anti-ATR, and anti-PCNA (A) or with anti-{gamma}H2AX (B). C, cells were treated as described in A and B and collected for comet assays as described in Materials and Methods. The levels of DNA strand breaks were quantitated and represented as damage scores on a scale from 0 to 4. Columns, mean (n = 3); bars, SE.

 
It was unknown whether the diminished ATR levels, which lead to an increase in H2AX activation, were accompanied by an increase of DNA strand breaks. Similarly for UV, the level of DNA breaks could also increase despite the drop in H2AX phosphorylation. To confirm these possibilities, DNA strand breaks were measured in a single-cell assay using alkaline comet analysis to detect both single-strand breaks and DSBs [for hedamycin, parallel neutral comet assays were also carried out and resulted in similar damage scores (at 5 nmol/L was 0.75 versus 0.8 in neutral and alkaline comet assay, respectively)]. In the absence of strand breaks, cellular DNA maintains a compact form during electrophoresis, whereas when DNA strand breaks are induced fragmented DNA migrates away from the nucleus and forms comet tails during electrophoresis. The level of drug-induced DNA strand breaks can be quantitated on a scale from 0 to 4 based on the length of the comet tails and expressed as an average level of DNA damage per cell as described in Materials and Methods (Fig. 2C). Following a 4-hour treatment, hedamycin-induced breaks are increased under ATR suppression condition, consistent with the increase of H2AX phosphorylation. In contrast, whereas UV-induced strand breaks also increased, H2AX phosphorylation decreased, suggesting that, unlike hedamycin, H2AX activation under conditions of ATR suppression does not reflect DNA strand breaks (Fig. 2C).

Hedamycin-Induced {gamma}H2AX Is Not Decreased in the Absence of ATM or DNA-PK nor in the Presence of Caffeine
Because suppression of ATR did not reduce phosphorylation of H2AX by hedamycin, we next examined whether H2AX phosphorylation would be altered in ATM and DNA-PK mutant cell lines. We also tested camptothecin, which is known to induce {gamma}H2AX in an ATR- and DNA-PK-dependent but ATM-independent manner to verify that our assay system produces similar findings (23). As expected, in response to camptothecin in ATM-deficient lymphoblasts, the level of H2AX phosphorylation was similar to that in wild-type cells (GM00536B). In contrast, hedamycin-induced phosphorylation of H2AX like in ATR-deficient cells increased in ATM-deficient compared with wild-type cells (Fig. 3A).



View larger version (51K):
[in this window]
[in a new window]
[Download PPT slide]
  		Figure 3. H2AX phosphorylation in response to hedamycin does not require ATM or DNA-PK nor is it suppressed by caffeine. (A) ATM-proficient cells (wild-type lymphoblasts, GM00536B) and ATM-deficient cells (AT lymphoblasts, GM01526E), and (B) DNA-PK-proficient cells (M059K) and DNA-PK-deficient cells (M059J) were exposed to either 5 nmol/L hedamycin for 4 h or 1 µmol/L camptothecin for 3 h. C, HCT116 cells were pretreated with 16 mmol/L caffeine for 30 min before treatment with hedamycin or camptothecin (CPT) as described in A and B. D, M059J cells were transfected with siRNA against ATR. At 24 h after transfection, cells were treated for hedamycin for 4 h. E, M059J cells were pretreated with 5 mmol/L caffeine for 30 min before treatment with hedamycin. Cell lysates were prepared and proteins were separated by SDS-PAGE and immunoblotted with anti-{gamma}H2AX and anti-PCNA.

 
In DNA-PK-deficient glioblastoma cells (M059J), as shown by others, camptothecin-induced phosphorylation of H2AX was reduced compared with wild-type cells (M059K; Fig. 3B). However, hedamycin-induced phosphorylation of H2AX was relatively similar in both cell lines.

It seems that, unlike other agents that arrest replication, any one of the three PIKKs does not reduce H2AX phosphorylation in response to hedamycin. To test whether H2AX phosphorylation would occur when the activity of multiple PIKKs are reduced, HCT116 cells were pretreated with 16 mmol/L caffeine for 30 minutes, which is sufficient to significantly reduce ATM and ATR activity before drug exposure (34). Figure 3C shows that, as expected, camptothecin-induced phosphorylation of H2AX was reduced by caffeine pretreatment. In contrast, hedamycin-induced phosphorylation of H2AX was increased compared with that of non-caffeine-treated cells. Similar results were also obtained when HCT116 cells were pretreated with wortmannin, which would effectively block ATM and DNA-PK activity (data not shown).

To further test how lack of PIKKs effects hedamycin-induced H2AX phosphorylation, DNA-PK-deficient M059J cells, which are known to also have low ATM levels, were transfected with a siRNA against ATR. At 24 hours after transfection, cells were treated with hedamycin for 4 hours. Figure 3D shows that hedamycin-induced H2AX phosphorylation was not decreased but increased under a condition where cells lacked or contained reduced levels of each of the PIKKs. Treatment of M059J cells with caffeine to reduce ATM/ATR activity also resulted in no reduction, but again an increase of hedamycin induced H2AX phosphorylation (Fig. 3E).

Induction of {gamma}H2AX by Hedamycin Can Occur in the Absence of Replication Fork Movement
Bulky DNA adducts, such as those induced by camptothecin and adozelesin, are generally converted into DSBs when active replication forks collide with drug adducts, leading to {gamma}H2AX formation (23, 24). To determine the dependency of hedamycin-induced {gamma}H2AX on active replication fork movement, HCT116 cells were pretreated with aphidicolin for 5 minutes to stall replication forks (as expected, [3H]thymidine incorporation was reduced by >90%; data not shown) and then incubated with either hedamycin or adozelesin for 1 hour or camptothecin for 3 hours. Subsequently, cell lysates were prepared and analyzed for H2AX phosphorylation by Western blotting. In agreement with other studies (23), the levels of H2AX phosphorylation induced by camptothecin and adozelesin in the presence of aphidicolin were significantly reduced 75% and 84%, respectively (Fig. 4A). In contrast, hedamycin-induced phosphorylation of H2AX was only moderately affected (30% reduction) in the presence of aphidicolin. We also examined {gamma}H2AX focalization under the same treatment condition (Fig. 4B shows representative images). Consistent with the Western blot results, adozelesin-induced {gamma}H2AX foci were substantially decreased in the presence of aphidicolin. In contrast, in hedamycin-treated cells, {gamma}H2AX foci were notably less dependent on replication fork movement. In total, these results show that hedamycin-induced strand breaks are not strongly dependent on DNA replication fork movement.



View larger version (29K):
[in this window]
[in a new window]
[Download PPT slide]
  		Figure 4. Effects of aphidicolin (APH) on the generation of {gamma}H2AX by hedamycin. A, Western blots showing H2AX phosphorylation. HCT116 cells were pretreated with 0.5 µmol/L aphidicolin for 5 min (which inhibits DNA synthesis by 90%) before treatment with 5 nmol/L hedamycin, 1 nmol/L adozelesin for 1 h, or 1 µmol/L camptothecin for 3 h. Cell lysates were prepared and proteins were separated by SDS-PAGE and immunoblotted with anti-{gamma}H2AX. B, immunofluorescence showing {gamma}H2AX focalization. Cells were treated as in A and immunostained as in Fig. 2B.

 
Hedamycin-Induced Strand Breaks Persist following Drug Removal
Replication-mediated DSBs are generally repaired after release from replication stress (35). To test if hedamycin-induced DNA strand breaks persist, hedamycin was removed from HCT116 cells following a 4-hour drug treatment and cells were incubated in drug-free medium for another 24 hours. Cells were then collected and subjected to alkaline comet analysis (whereas alkaline comet analysis provides a sensitive way to detect both single-strand breaks and DSBs, induction of DSBs by neutral comets revealed similar profiles). As shown in Fig. 5A and B, more extensive comets appeared at all levels of hedamycin treatments compared with 4-hour treatments (data not shown). For example, the DNA strand break score increased from 0.2 (4 hours) to 1.5 (24 hours) at 0.25 nmol/L and from 0.8 (4 hours) to 2.1 (24 hours) at 1 nmol/L. Notably, the extent of strand breaks reached a peak at 1 nmol/L and actually declined by 5 nmol/L.



View larger version (38K):
[in this window]
[in a new window]
[Download PPT slide]
  		Figure 5. Accumulation of DNA strand breaks by hedamycin at 24 h after drug removal. HCT116 cells were treated with hedamycin for 4 h and further incubated in drug-free medium for 24 h. Cells were collected for comet assays as described in Materials and Methods. A, nuclei DNA was stained with ethidium bromide. B, levels of DNA strand breaks were quantitated and represented as damage scores on a scale from 0 to 4. Points, mean (n = 3); bars, SE.

 
Hedamycin Induces Chromosome Aberrations
Hedamycin-induced DNA strand breaks seem to persist in interphase nuclear DNA. It is possible that such unrepaired strand breaks would lead to chromosome aberrations in mitotic cells (36). To test if hedamycin-induced DNA damage is associated with chromosomal aberrations, hedamycin was removed from HCT116 cells after a 4-hour drug treatment and cells were incubated in drug-free medium for another 24 hours. At 2 hours before harvest, the mitotic cell population was enriched by the addition of colcemid and then analyzed for chromosomal aberrations using spectral karyotyping analysis. Images reveal that hedamycin induces a significant amount of chromosomal misjoining and fragmentation events compared with untreated cells (Fig. 6). The most frequent types of chromosomal aberration induced by hedamycin were chromatid/chromosome breaks, gaps (Fig. 6A, c-f, arrowheads), end joining (Fig. 6A, c and d, open arrows), and nonhomologous recombination forming quadriradial chromosomes (Fig. 6A, c-f, arrows). At 0.25 nmol/L, the major type of aberrations was end-to-end association.



View larger version (48K):
[in this window]
[in a new window]
[Download PPT slide]
  		Figure 6. Hedamycin induces chromosome aberrations in mitotic cells. A, metaphase chromosomes of HCT116 cells in normal cells (a and b) and hedamycin-treated cells (c-f): 4',6-diamidino-2-phenylindole banding (a, c, and e) and spectral karyotyping analysis (b, d, and f). Normal cells contain model chromosome numbers of 45 and specific translocation markers (filled arrows). Some untreated cells show chromatid gaps (like the one shown in a; open arrow). Many of the hedamycin-treated cells containing nonhomologous end-to-end chromatid association forming quadriradial chromosomes (arrows; c-f) also contain chromatid/chromosome gaps and breaks, which reflect repair failure (arrowheads). Some quadrivalent chromosomes display incomplete end-to-end chromatid associations leaving some chromatid ends unfused (open arrow in c and d). B, metaphase chromosome undergoing chromatid/chromosome breaks (a and b) and extreme fragmentation (c and d). Arrows, some of the prominent chromosomes damaged by breaks.

 
Table 1 summarizes the effects of hedamycin on the numbers and types of chromosomal aberrations. Chromosomal aberrations increased with drug concentration, and profound chromosomal aberrations occurred at 1 to 2.5 nmol/L where cells progressing through S phase were slowed and the highest levels of interphase DNA breaks were detected by comet assays. Although the magnitude of DNA damage reached near saturation at >1 nmol/L, it is of special interest to note that only 21% of mitotic cells contained chromosome aberrations. However, when the drug concentration was increased to 1 to 2.5 nmol/L, >60% of the cells with damaged chromosomes contained gaps/breaks and fragmentation (Fig. 6B; Table 1).


View this table:
[in this window]
[in a new window]

  		Table 1. Analysis of chromosome aberrations following a 4-hour hedamycin treatment in HCT116 cells

 
Ku80 Forms Nuclear Foci in Response to Hedamycin
The significant numbers of NHEJ events detected by spectral karyotyping analysis suggested that proteins involved in NHEJ repair might be recruited to the site of hedamycin-induced DSBs. Ku80 is a DNA end-binding protein that preferentially interacts with chromosomal breaks (e.g., ionizing radiation induced), which not only protect DNA ends from nuclease attack but also is an essential component of the NHEJ machinery used in DSB repair (37).

Because Ku80 is an abundant protein that distributes in both nuclear and cytosolic fractions, detection of distinct nuclear foci requires detergent extraction (38, 39). HCT116 cells treated with hedamycin were extracted to remove nucleoplasmic protein while retaining detergent-resistant chromatin-bound Ku80. Localization of Ku80 was examined using immunofluorescence microscopy. Figure 7 shows representative images. In untreated control cells, Ku80 aggregated in the nucleolus but did not tightly associate with chromatin. Following 1 nmol/L hedamycin treatment, a concentration that induced significant NHEJ events, most of the Ku80 was redistributed and tightly bound to chromatin while being lost from the nucleolus. Like {gamma}H2AX, H2AX, the Ku80 foci persisted at the DNA damage site because it was also observed at both the 4-hour treatment and even at 24 hours after drug removal.



View larger version (69K):
[in this window]
[in a new window]
[Download PPT slide]
  		Figure 7. Hedamycin-induced DNA strand breaks are associated with Ku80 foci formation. HCT116 cells were treated with 1 nmol/L hedamycin for 1 or 4 h. Cells were fixed and immunostained with anti-Ku80 and anti-mouse immunoglobulin antibody conjugated with Texas red. Nuclei were stained with Hoechst 33258.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
Recent studies suggest that PIKK-regulated DNA damage checkpoint responses to DNA adducts may be triggered by the subsequent generation of single-strand DNA or DNA breaks resulting from sustained DNA replication arrest (7, 40). This study revealed that hedamycin is similar to other treatments, such as UV or camptothecin, which induce bulky DNA lesions accompanied by DNA strand breaks. Initially, UV and camptothecin bulky DNA lesions activate chk1 via ATR and subsequently, also through ATR, activate chk2 to cope with strand breaks (41, 42). Similarly, hedamycin under conditions that induce strand breaks also induces checkpoint protein phosphorylation, such as chk1 and chk2 (25), suggesting that DNA strand breaks likely contribute to the DNA damage responses. Following DSB induction, H2AX undergoes phosphorylation at a conserved PIKK motif before focalization at DNA break sites (43).

Replication stress induced by diverse genotoxins, including hydroxyurea, camptothecin, and monofunctional alkylating agents, such as adozelesin, and methylmethanesulfonate all generate DSBs based on induction of the DNA damage response protein {gamma}H2AX (17, 23, 24). Western and immunofluorescence microscopy analyses revealed that {gamma}H2AX also appears in hedamycin-treated cells and increases in parallel with DNA breaks, suggesting that hedamycin-DNA adducts are also converted to DSBs (Fig. 1).

Typically, H2AX is phosphorylated by ATR in response to replication-mediated DSBs, such as those induced by UV and camptothecin (33). However, despite the many similarities to these agents, hedamycin-induced DSBs result in extensive phosphorylation of H2AX when ATR is suppressed. Hedamycin-induced H2AX phosphorylation actually increases under ATR suppression conditions, which is in striking contrast to many other genotoxins, such as UV, hydroxyurea, and camptothecin, which all show significant decreases. Moreover, for hedamycin, the assessment of DNA strand breaks by comet assay in ATR-reduced cells (Fig. 2C) parallels the increase in {gamma}H2AX. Intriguingly, UV-induced breaks, as determined directly by comet analysis, also increased in ATR-reduced cells (Fig. 2C), although we observed like others a marked reduction of H2AX phosphorylation (Fig. 2B). Although H2AX phosphorylation is widely used as a measure of induction of DSB, our analysis indicates its limitations. For hedamycin, DSBs and H2AX are concordant, but when ATR is required to activate H2AX, such as the case with UV, it is difficult to distinguish whether decreased {gamma}H2AX stems from loss of signaling or truly reflects diminished DSBs. Overall, when ATR is defective, replication may be less likely to stop before a collision with a DNA lesion and/or stalled replication fork collapse, both of which can elevate DNA breaks (44).

Loss of other individual PIKKs, ATM and DNA-PK, and suppression of PIKK activity by high concentrations of caffeine also do not decrease hedamycin-induced {gamma}H2AX (Figs. 2 and 3). Currently, we are evaluating whether kinases other than ATM, ATR, and DNA-PK, such as the recently described hSmg-1 (ATX), plays a role in regulating hedamycin.

Characteristically, replication is required for DSB generation, such as with camptothecin and adozelesin, because breaks are generated as a consequence of collisions between sites of stalled replication and advancing forks (23, 24). In these cases, the addition of aphidicolin and inhibitor of DNA chain elongation nearly eliminates H2AX phosphorylation and its focalization. Although hedamycin is unlike these genotoxic agents in that ATR is not the predominant PIKK responding to DNA adduct, it was unexpected that H2AX phosphorylation and focalization were also rather independent of fork movement (Fig. 4). Consistent with the finding that aphidicolin does not significantly prevent hedamycin-induced {gamma}H2AX, the strong intensity of {gamma}H2AX foci in >90% of hedamycin-treated cells after 4-hour treatment suggests that hedamycin-induced DSBs, unlike camptothecin and adozelesin, are not limited to S-phase cells. However, treatment of G1-S synchronization cells with hedamycin resulted in an elevation of strand breaks and {gamma}H2AX compared with asynchronous cultures (data not shown), indicating perhaps that ongoing replication can still contribute to the level of DNA breaks.

Our previous study reported that hedamycin-induced DNA damage checkpoint activation, including chk1, chk2, and p53 phosphorylation, is maintained for at least 24 hours after drug removal (25), suggesting that hedamycin lesions may be persistent. The high levels of hedamycin-induced strand breaks (Fig. 5) observed 24 hours after drug removal are consistent with both the DNA lesions being refractory to removal and a sustained DNA damage checkpoint response that triggers a significant decrease in S-phase progression. Interestingly, the extent of strand breaks somewhat diminished as the drug concentration was raised from 1 to 5 nmol/L (Fig. 5). Previously, we showed that 1 nmol/L hedamycin is not sufficient to promote cdc25A degradation after a 4-hour treatment (25), allowing cells to enter S phase and replicate damaged DNA, which would likely contribute to strand breaks. By contrast, hedamycin at 5 nmol/L promotes cdc25A degradation, blocking entry into S phase to minimize the potential of generating breaks due to replication of damaged DNA. Also, damaged DNA in cells progressing through S phase would be subject to collision with adducts, stalled replication fork collapse, and induction of repair intermediates under conditions where many of the cells cannot complete the transit out of S phase (21, 35). In contrast, cells treated at 5 nmol/L hedamycin never exit from the G1 arrest and strand breaks would more likely result from accumulation of repair intermediates.

Possibly, hedamycin-induced apoptotic responses could also contribute to DNA strand breaks (25). However, apoptotic contribution to DNA breaks likely is minimal. First, significant levels of DNA strand breaks are observed at concentrations that induce barely detectable levels of apoptosis. Second, cells displaying {gamma}H2AX foci were not undergoing apoptosis based on microscopic examination. Moreover, {gamma}H2AX does not form foci in apoptotic cells (45). Third, fragmented DNA in the form of DNA laddering that is associated with apoptosis was not observed when DNA strand breaks were analyzed using pulse-field electrophoresis.4

The persistently elevated levels of DNA damage observed in interphase cells may translate into chromosomal aberrations (46). Elevated chromosomal aberrations were observed in those hedamycin-treated cells able to progress into metaphase. Among the chromosomal aberrations induced following moderate DNA damage (e.g., Fig. 6A, a-d), hedamycin induces a predominance of NHEJ events, indicating that NHEJ was a mechanism activated in the repair of hedamycin-induced DSBs. Notably, hedamycin produces many quadriradial chromosomes, composed from two to three chromosomes, which had been reported only in bifunctional DNA cross-linker–treated cells (47). Collectively, these results imply the following: (a) that some of the initial damage induced by hedamycin was effectively repaired during the cell cycle phase, which could promote cell entry into mitosis; (b) that end-to-end association forming quadriradial chromosomes stem from some form of repair response; and (c) that excessive DNA damage can overwhelm the capacity of repair systems resulting in gaps/breaks and fragmentation of chromosomes.

Evidence supporting a role for NHEJ in processing hedamycin-induced DSBs is that enhanced Ku80 relocalization and focalization in tight-binding chromatin-bound complexes was detected following short (1 hour) and long-term (24 hours) hedamycin treatments (Fig. 7). Ku80 is a DSB sensor, which subsequently conducts DSB repair through activation of DNA-PK and recruitment of ligase IV (48). Once repair is complete, tight binding of Ku80 should diminish. Notably, for hedamycin, Ku80 foci continued to increase over time and associate with drug-induced DSBs, consistent with the idea that the misjoined chromosomes observed in metaphase cells could have resulted from a failure to complete NHEJ. Alternatively, Ku80/DNA-PK can activate a p53-mediated apoptotic pathway when repair failure leads to persistence of DSBs (49), yet hedamycin does not induce significant levels of apoptosis (25).

Hedamycin induces DNA damage responses that are atypical of most genotoxic agents examined to date. Studies are currently under way to assess how hedamycin interferes with DNA replication and to determine whether its mechanism of disruption of replication relates to its unique DNA damage response.


   Acknowledgments
 
We thank Dr. Gokul Das for the advice and comments on the article.


   Footnotes
 
Grant support: NIH grants CA77491 and CA16056 (T.A. Beerman).

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.

3 M.G.J. Hartl (http://www.ucc.ie/ucc/depts/zoology/biomasstox). Back

4 J. Dziegielewski and T.A. Beerman, unpublished results. Back

Received 2/22/05; revised 5/17/05; accepted 6/ 1/05.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

  1. Norbury CJ, Hickson ID. Cellular responses to DNA damage. Annu Rev Pharmacol Toxicol 2001;41:367–401.[CrossRef][Medline]
  2. Lundin C, Erixon K, Arnaudeau C, et al. Different roles for nonhomologous end joining and homologous recombination following replication arrest in mammalian cells. Mol Cell Biol 2002;22:5869–78.[Abstract/Free Full Text]
  3. Pourquier P, Pommier Y. Topoisomerase I-mediated DNA damage. Adv Cancer Res 2001;80:189–216.[Medline]
  4. Shiloh Y. ATM and ATR: networking cellular responses to DNA damage. Curr Opin Genet Dev 2001;11:71–7.[CrossRef][Medline]
  5. Michel B, Ehrlich SD, Uzest M. DNA double-strand breaks caused by replication arrest. EMBO J 1997;16:430–8.[CrossRef][Medline]
  6. Weinert T. DNA damage and checkpoint pathways: molecular anatomy and interactions with repair. Cell 1998;94:555–8.[CrossRef][Medline]
  7. Shechter D, Costanzo V, Gautier J. Regulation of DNA replication by ATR: signaling in response to DNA intermediates. DNA Repair (Amst) 2004;3:901–8.
  8. Laiho M, Latonen L. Cell cycle control, DNA damage checkpoints and cancer. Ann Med 2003;35:391–7.[CrossRef][Medline]
  9. Rogakou EP, Pilch DR, Orr AH, Ivanova VS, Bonner WM. DNA double-stranded breaks induce histone H2AX phosphorylation on serine 139. J Biol Chem 1998;273:5858–68.[Abstract/Free Full Text]
  10. Rogakou EP, Boon C, Redon C, Bonner WM. Megabase chromatin domains involved in DNA double-strand breaks in vivo. J Cell Biol 1999;146:905–16.[Abstract/Free Full Text]
  11. D'Amours D, Jackson SP. The Mre11 complex: at the crossroads of DNA repair and checkpoint signalling. Nat Rev Mol Cell Biol 2002;3:317–27.[CrossRef][Medline]
  12. Paull TT, Rogakou EP, Yamazaki V, et al. A critical role for histone H2AX in recruitment of repair factors to nuclear foci after DNA damage. Curr Biol 2000;10:886–95.[CrossRef][Medline]
  13. Smith R, Kao GD. Pictures, progress, and perplexities: the immediate cell biological effects of ionizing radiation. Cancer Biol Ther 2004;3:602–7. Epub 2004 July 21.[Medline]
  14. Pasero P, Shimada K, Duncker BP. Multiple roles of replication forks in S phase checkpoints: sensors, effectors and targets. Cell Cycle 2003;2:568–72.[Medline]
  15. Larner JM, Lee H, Little RD, et al. Radiation down-regulates replication origin activity throughout the S phase in mammalian cells. Nucleic Acids Res 1999;27:803–9.[Abstract/Free Full Text]
  16. Tercero JA, Diffley JF. Regulation of DNA replication fork progression through damaged DNA by the Mec1/Rad53 checkpoint. Nature 2001;412:553–7.[CrossRef][Medline]
  17. Ward IM, Chen J. Histone H2AX is phosphorylated in an ATR-dependent manner in response to replicational stress. J Biol Chem 2001;276:47759–62.[Abstract/Free Full Text]
  18. Burma S, Chen BP, Murphy M, Kurimasa A, Chen DJ. ATM phosphorylates histone H2AX in response to DNA double-strand breaks. J Biol Chem 2001;276:42462–7.[Abstract/Free Full Text]
  19. Abraham RT. Cell cycle checkpoint signaling through the ATM and ATR kinases. Genes Dev 2001;15:2177–96.[Free Full Text]
  20. Giannattasio M, Lazzaro F, Longhese MP, Plevani P, Muzi-Falconi M. Physical and functional interactions between nucleotide excision repair and DNA damage checkpoint. EMBO J 2004;23:429–38.[CrossRef][Medline]
  21. Paulovich AG, Toczyski DP, Hartwell LH. When checkpoints fail. Cell 1997;88:315–21.[CrossRef][Medline]
  22. Strumberg D, Pilon AA, Smith M, et al. Conversion of topoisomerase I cleavage complexes on the leading strand of ribosomal DNA into 5'-phosphorylated DNA double-strand breaks by replication runoff. Mol Cell Biol 2000;20:3977–87.[Abstract/Free Full Text]
  23. Furuta T, Takemura H, Liao ZY, et al. Phosphorylation of histone H2AX and activation of Mre11, Rad50, and Nbs1 in response to replication-dependent DNA double-strand breaks induced by mammalian DNA topoisomerase I cleavage complexes. J Biol Chem 2003;278:20303–12.[Abstract/Free Full Text]
  24. Liu JS, Kuo SR, Melendy T. Comparison of checkpoint responses triggered by DNA polymerase inhibition versus DNA damaging agents. Mutat Res 2003;532:215–26.[Medline]
  25. Tu LC, Melendy T, Beerman TA. DNA damage responses triggered by a highly cytotoxic monofunctional DNA alkylator, hedamycin, a pluramycin antitumor antibiotic. Mol Cancer Ther 2004;3:577–85.[Abstract/Free Full Text]
  26. Hansen M, Yun S, Hurley L. Hedamycin intercalates the DNA helix and, through carbohydrate-mediated recognition in the minor groove, directs N7-alkylation of guanine in the major groove in a sequence-specific manner. Chem Biol 1995;2:229–40.[CrossRef][Medline]
  27. Casper AM, Nghiem P, Arlt MF, Glover TW. ATR regulates fragile site stability. Cell 2002;111:779–89.[CrossRef][Medline]
  28. Collins AR, Dusinska M, Horska A. Detection of alkylation damage in human lymphocyte DNA with the comet assay. Acta Biochim Pol 2001;48:611–4.[Medline]
  29. Cortes F, Morgan WF, Wolff S. Effect of exogenous thymidine on sister-chromatid exchange frequency in Chinese hamster ovary cells with bromodeoxyuridine- and chlorodeoxyuridine-substituted chromosomes. Mutat Res 1987;192:277–82.[CrossRef][Medline]
  30. Cleaver JE, Laposa RR, Limoli CL. DNA replication in the face of (in)surmountable odds. Cell Cycle 2003;2:310–5.[Medline]
  31. Pommier Y, Redon C, Rao VA, et al. Repair of and checkpoint response to topoisomerase I-mediated DNA damage. Mutat Res 2003;532:173–203.[Medline]
  32. Rogakou EP, Nieves-Neira W, Boon C, Pommier Y, Bonner WM. Initiation of DNA fragmentation during apoptosis induces phosphorylation of H2AX histone at serine 139. J Biol Chem 2000;275:9390–5.[Abstract/Free Full Text]
  33. Durocher D, Jackson SP. DNA-PK, ATM, ATR. as sensors of DNA damage: variations on a theme? Curr Opin Cell Biol 2001;13:225–31.[CrossRef][Medline]
  34. Cortez D. Caffeine inhibits checkpoint responses without inhibiting the ataxia-telangiectasia-mutated (ATM) and ATM- and Rad3-related (ATR) protein kinases. J Biol Chem 2003;278:37139–45.[Abstract/Free Full Text]
  35. McGowan CH. Running into problems: how cells cope with replicating damaged DNA. Mutat Res 2003;532:75–84.[Medline]
  36. Iliakis G, Wang H, Perrault AR, et al. Mechanisms of DNA double strand break repair and chromosome aberration formation. Cytogenet Genome Res 2004;104:14–20.[CrossRef][Medline]
  37. Lieber MR, Ma Y, Pannicke U, Schwarz K. Mechanism and regulation of human non-homologous DNA end-joining. Nat Rev Mol Cell Biol 2003;4:712–20.[CrossRef][Medline]
  38. Koike M, Ikuta T, Miyasaka T, Shiomi T. Ku80 can translocate to the nucleus independent of the translocation of Ku70 using its own nuclear localization signal. Oncogene 1999;18:7495–505.[CrossRef][Medline]
  39. Fewell JW, Kuff EL. Intracellular redistribution of Ku immunoreactivity in response to cell-cell contact and growth modulating components in the medium. J Cell Sci 1996;109:1937–46.[Abstract]
  40. Nyberg KA, Michelson RJ, Putnam CW, Weinert TA. Toward maintaining the genome: DNA damage and replication checkpoints. Annu Rev Genet 2002;36:617–56.[CrossRef][Medline]
  41. Chen Y, Sanchez Y. Chk1 in the DNA damage response: conserved roles from yeasts to mammals. DNA Repair (Amst) 2004;3:1025–32.
  42. 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]
  43. Modesti M, Kanaar R. DNA repair: spot(light)s on chromatin. Curr Biol 2001;11:R229–32.[CrossRef][Medline]
  44. Heffernan TP, Simpson DA, Frank AR, et al. An ATR- and Chk1-dependent S checkpoint inhibits replicon initiation following UVC-induced DNA damage. Mol Cell Biol 2002;22:8552–61.[Abstract/Free Full Text]
  45. Huang X, Okafuji M, Traganos F, et al. Assessment of histone H2AX phosphorylation induced by DNA topoisomerase I and II inhibitors topotecan and mitoxantrone and by the DNA cross-linking agent cisplatin. Cytometry A 2004;58A:99–110.[Medline]
  46. Bassing CH, Alt FW. The cellular response to general and programmed DNA double strand breaks. DNA Repair (Amst) 2004;3:781–96.
  47. Matsumoto L, Kurek K, Larocque K, et al. Biological effects of a bifunctional DNA crosslinker. I. Generation of triradial and quadriradial chromosomes. Mutat Res 1999;426:79–87.[Medline]
  48. Downs JA, Jackson SP. A means to a DNA end: the many roles of Ku. Nat Rev Mol Cell Biol 2004;5:367–78.[CrossRef][Medline]
  49. Bree RT, Neary C, Samali A, Lowndes NF. The switch from survival responses to apoptosis after chromosomal breaks. DNA Repair (Amst) 2004;3:989–95.




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 Google Scholar
Google Scholar
Right arrow Articles by Tu, L. C.
Right arrow Articles by Beerman, T. A.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Tu, L. C.
Right arrow Articles by Beerman, T. A.

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