Vol. 2, 41-47, January 2003
Molecular Cancer Therapeutics
© 2003 American Association for Cancer Research
Induction of DNA Damage Responses by Adozelesin Is S Phase-specific and Dependent on Active Replication Forks1
Jen-Sing Liu2,
Shu-Ru Kuo2,
Terry A. Beerman and
Thomas Melendy3
Departments of Microbiology and Biochemistry and the Witebsky Center for Microbial Pathogenesis and Immunology, University at Buffalo, SUNY, School of Medicine and Biomedical Sciences, Buffalo, New York 14214 [J-S. L., S-R. K., T. M.], and Department of Pharmacology and Therapeutics, Roswell Park Cancer Institute, Buffalo, New York 14263 [T. A. B.]
 |
Abstract
|
|---|
Adozelesin is an alkylating minor groove DNA binder that is capable of rapidly inhibiting DNA replication in treated cells through a trans-acting mechanism and preferentially arrests cells in S phase. It has been shown previously that in cells treated with adozelesin, replication protein A (RPA) activity is deficient, and the middle subunit of RPA is hyperphosphorylated. The adozelesin-induced RPA hyperphosphorylation can be blocked by the replicative DNA polymerase inhibitor, aphidicolin, suggesting that adozelesin-triggered cellular DNA damage responses require active DNA replication forks. These data imply that cellular DNA damage responses to adozelesin treatment are preferentially induced in S phase. Here, we show that RPA hyperphosphorylation, RPA intranuclear focalization, and 
-H2AX intranuclear focalization induced by adozelesin treatment are all dependent on DNA replication fork progression, and focalization is only induced in S phase cells. These findings are similar to those seen with the S phase-specific DNA-damaging agent, camptothecin. Conversely, all three DNA damage responses are independent of either S phase or replication fork progression when induced by treatment with the DNA strand scission agent, C-1027. Furthermore, we demonstrate that adozelesin-induced RPA and -H2AX intranuclear foci appear to colocalize within the nuclei of S phase cells.
|
Introduction
|
|---|
The anticancer agent adozelesin is an analogue of CC-1065, a CPI3 isolated from Streptomyces zelensis. Adozelesin binds to the minor groove of A/T-rich DNA sequences and alkylates the N3 of adenines at the 3'-end of its binding sites (1, 2). This results in the formation of adozelesin:DNA adducts, which leads to the inhibition of both cellular and viral DNA replication and a S phase cell cycle arrest (3, 4). Previously published results show that the inhibition of DNA replication by adozelesin occurs through a trans-acting mechanism. The trans-acting replication factor that is inactivated on treatment with adozelesin has been identified as RPA (5). RPA is the major eukaryotic single-strand DNA binding protein. This heterotrimeric protein (Mr 70,000; 32,000; and 14,000) is essential for DNA replication and plays critical roles in DNA repair and recombination (6, 7).
The NH2-terminal domain of the Mr 32,000 subunit of RPA (RPA32) has been shown to become hyperphosphorylated during S phase of the cell cycle. RPA32 hyperphosphorylation is also induced in response to DNA damage (6, 7). Shao et al. (8) reported that induction of RPA32 hyperphosphorylation by radiation or CPT could be prevented by pretreating cells with the replicative DNA polymerase inhibitor, aphidicolin. Similar results have also been observed in UV-irradiated and adozelesin-treated cells (5, 9, 10). These findings suggested that RPA32 hyperphosphorylation in response to DNA damage might be dependent on the active passage of DNA replication fork. However, when cells were treated with enediyne C-1027, the induction of RPA32 hyperphosphorylation was found to be completely resistant to aphidicolin (11). This suggests the presence of both replication-dependent and -independent mechanisms for RPA32 hyperphosphorylation.
CPT, a topoisomerase I inhibitor, is known to selectively induce S phase DNA damage checkpoints and causes DSBs only when there is DNA replication fork movement (12). This may explain why RPA32 hyperphosphorylation induced by CPT is dependent on replication fork progression. It is also understandable how C-1027, a DNA scission agent that directly binds and breaks one or both strands of DNA (11, 13, 14), can induce RPA32 hyperphosphorylation independent of replication fork movement. However, it is unclear why replication fork progression would be required for RPA32 hyperphosphorylation induced by agents like adozelesin, which directly damage DNA.
In S phase cells, a small portion of RPA becomes tightly associated with the nuclear matrix to form intranuclear foci, which were found to correspond with sites of DNA replication (15–18). RPA focalization was also observed in UV- or -irradiated cells, and these RPA foci colocalized with other DNA repair factors (19–23). It has not been demonstrated whether DNA damage-induced RPA focalization also has the same dependency on DNA replication fork progression as RPA32 hyperphosphorylation.
In this study, we study how treatment of human cells with these three DNA-damaging agents (CPT, C-1027, and adozelesin; Fig. 1) triggers these early cellular DNA damage responses and the role that S phase and DNA replication play in these responses. We have used relatively high levels of these three agents over short time frames to focus primarily on the early responses to DNA damage. Specifically, we addressed the question of whether adozelesin triggers these DNA damage responses in an S phase-specific manner. We examined phosphorylation of RPA and formation of RPA and -H2AX (histone H2AX phosphorylated at serine 139) foci in response to adozelesin, CPT, and C-1027. The induction of -H2AX foci is known as an early cellular response to either DSBs or replicational stress (for review, see Ref. 24). CPT and C-1027 were used as examples of S phase-specific and nonspecific DNA-damaging agents, respectively. Our results show that like CPT, adozelesin-induced RPA32 hyperphosphorylation and -H2AX and RPA focalization are S phase specific and require active DNA replication fork progression.
|
Materials and Methods
|
|---|
Chemicals and Antibodies.
CPT (10 mM, dissolved in DMSO), aphidicolin (1 mg/ml, dissolved in ethanol), and DAPI were purchased from Sigma Chemical Co. (St. Louis, MO). Adozelesin, generously supplied by Pharmacia Upjohn Co. (Kalamazoo, MI), was dissolved in dimethylacetamide (2 mg/ml) and further diluted in DMSO before its addition into culture medium. C-1027, a gift from Taiho Pharmaceuticals Co. Ltd (Saitama, Japan), was diluted in water. Both BUdR and anti-BUdR monoclonal antibody were purchased from BD PharMingen. Monoclonal antibody against -H2AX was purchased from Upstate Biotechnology. The monoclonal antibody against RPA32 was described by Din et al. (25), and polyclonal antibody was raised against an MBP-RPA32 fusion protein (26) and affinity-purified against His-tagged RPA32. Fluorescein-conjugated goat antimouse and Alexa 568-conjugated goat-antirabbit antibodies were purchased from Vector Laboratories, Inc. and Molecular Probes, respectively.
Cell Line and Cell Culture.
Monolayer cultured HeLa cells (American Type Culture Collection) maintained in DMEM with 10% fetal bovine serum were grown in two-well chamber slides (Nalge Nunc International) for immunostaining studies or in 60-mm plates for immunoblotting assays. Asynchronous cells were incubated with or without 2.5 µM aphidicolin for 1 h before and throughout the treatment. DNA-damaging agents were then added at the indicated concentrations. For S phase block, the cells were treated with 25 µM aphidicolin for 16–18 h. DNA-damaging agents were then added to the cell cultures directly or 1 h after changing to fresh medium. To monitor newly synthesized DNA, 20 mM BUdR were added to the culture medium for 30 min. After being replaced with fresh medium, these cells were then mock treated or treated with the indicated DNA-damaging agents.
Indirect Immunofluorescent Staining.
The procedure for indirect immunofluorescent staining has been described previously (27). Briefly, after DNA-damaging treatments, cells were permeabilized and washed with 0.5% Triton X-100 in PBS to remove the free nucleosolic proteins, followed by paraformaldehyde (3% in PBS) fixation. The extraction-resistant RPA and -H2AX were stained with antigen-specific primary antibodies and fluorescein- or Alexa 568-conjugated secondary antibodies. DAPI (2 mM) was used to stain DNA. In experiments with pulse-labeled BUdR, a two-step staining protocol was used (17). Antigen-purified polyclonal RPA32 antibody and Alexa 568-conjugated goat-antirabbit antibody were first used to stain RPA. The bound antibodies were then fixed in situ with 3% paraformaldehyde in PBS at room temperature for 15 min. This was followed by acid denaturation of the DNA. BUdR-specific monoclonal antibody and fluorescein-conjugated antimouse antibody were then used to stain the newly synthesized DNA. RPA and -H2AX foci and BUdR incorporation were examined using an Olympus BX40 microscope with a SPOT-RT digital camera and software. Adobe PhotoShop was used for image processing and printing. The percentages of RPA-positive cells were calculated based on the number of DAPI-stained nuclei that were positive for fluorescein staining. For each preparation, the number was calculated using 200–300 cells and rounded to the nearest whole number.
Western Blot Hybridization.
Mock-treated or DNA-damaging agent-treated cells (1 x 106) were washed with cold PBS and lysed directly in SDS sample buffer [20 mM Tris-HCl (pH 7.5), 2% SDS, and 1 M 2-mercaptoethanol]. Total protein from an equal number of cells (
2 x 104) was resolved by electrophoresis on a 12.5% (w/v) SDS-PAGE and transferred to Hybond-P membrane using NovaBlot (Amersham Pharmacia Biotech) as per the manufacturer’s instructions. The membranes were probed with a monoclonal antibody against RPA32 and peroxidase-conjugated goat antimouse IgG (Pierce). The membranes were then treated with Supersignal enhanced chemiluminescent reagent (Pierce) and exposed to X-ray film.
|
Results
|
|---|
DNA Damage-induced RPA and -H2AX Focus Formation.
The monolayer cultured HeLa cells used in this study showed similar patterns of RPA32 hyperphosphorylation as human 293 cells from results published previously (5, 11). RPA32 hyperphosphorylation is triggered by treatment with as little as 1 nM adozelesin, 1 µM CPT, or 0.1 nM C-1027, with increasing levels of drug resulting in increasing levels of hyperphosphorylated RPA32 (Refs. 3 and 9 and data not shown). Aphidicolin pretreatment blocks RPA32 hyperphosphorylation induced by CPT or adozelesin at all levels tested (Fig. 2, Lanes 3–6, and data not shown) but not that induced by C-1027 (Fig. 2, Lanes 7 and 8). These results suggest that the activation of the kinase responsible for RPA32 hyperphosphorylation in cells treated with CPT or adozelesin requires DNA synthesis.
View larger version (15K):
[in this window]
[in a new window]
[Download PPT slide]
|
Fig 2. DNA damage-induced RPA32 hyperphosphorylation in asynchronous cells. Exponentially growing HeLa cells were either mock treated (Lanes 1 and 2) or treated for 2 h with 50 µM CPT (Lanes 3 and 4), 40 nM adozelesin (Adozel; Lanes 5 and 6), or 1 nM C-1027 (Lanes 7 and 8), either with (even number lanes) or without (odd number lanes) pretreating the cells with 2.5 µM aphidicolin for 1 h (aphidicolin treatment maintained throughout DNA-damaging treatment). Total proteins were separated by SDS-PAGE and immunoblotted for RPA32. Migration of RPA32 and hyperphosphorylated RPA32 (RPA32-Pi) are indicated on the right.
|
|
The effects of aphidicolin on DNA damage-induced RPA and -H2AX focus formation were then tested. Conditions for causing DNA damage in the presence or absence of aphidicolin were the same as that used in Fig. 2. After drug treatment, cells were washed with 0.5% Triton X-100 in PBS to permeabilize the nucleus and wash away the loosely associated nucleosolic proteins before fixation and immunostaining. A monoclonal antibody specific to -H2AX and polyclonal antibody against RPA32 were used as primary antibodies in indirect immunofluorescent staining. The nuclei of mock-treated cells contain very low levels of extraction-resistant RPA or -H2AX (Fig. 3A1–A3). Treatment of cells with any of the three DNA-damaging drugs induced high levels of intranuclear detergent-resistant RPA and -H2AX (Fig. 3, B–D, Lanes 1–3). However, the percentage of cells that contained RPA or -H2AX foci varied. Treatment of cells with CPT or adozelesin induced both RPA and -H2AX foci in 38% of cells (Fig. 3, B and C, Lanes 1–3). In contrast, the vast majority of C-1027-treated cells (92%) contained high levels of RPA and -H2AX foci (Fig. 3D, Lanes 1–3). One hour of aphidicolin pretreatment eliminated the majority of RPA and -H2AX staining in cells treated with either CPT or adozelesin (Fig. 3, B and C, Lanes 4–6). In C-1027-treated cells, aphidicolin had virtually no effect on RPA or -H2AX focus formation (Fig. 3D, Lanes 4–6). These results demonstrate that DNA damage-induced RPA and -H2AX focalization, as well as RPA32 hyperphosphorylation, can be either sensitive (for CPT and adozelesin) or resistant (for C-1027) to aphidicolin.
Because RPA and -H2AX foci appeared in the same cells after treatment with either adozelesin or CPT, the possibility of colocalization of these two signals was examined. As shown in Fig. 4, under higher magnification, RPA and -H2AX foci show very similar patterns in cells treated with adozelesin. The merged image of the RPA and -H2AX foci shows these patterns to be highly similar, suggesting a high degree of colocalization.
Adozelesin-induced RPA Focus Formation in S Phase Cells.
The ability of aphidicolin to block RPA focus formation and hyperphosphorylation in cells treated with adozelesin suggested that the adozelesin-triggered changes in RPA are S phase-specific responses. To address this possibility, asynchronous cells were pulse labeled with BUdR to identify cells in S phase. After several washes with fresh medium, cells were either mock treated or treated with DNA-damaging agent for 1 h and then stained for both RPA and BUdR (see "Materials and Methods"). In the absence of DNA-damaging agents, 35% of the cells show both BUdR incorporation and weak but clearly detectable levels of RPA focalization (Fig. 5A). This result suggests that those cells with weak RPA staining nuclei are in fact in S phase. As in Fig. 3, treatment with adozelesin or CPT results in strong RPA staining in a fraction (35%) of asynchronous cells (Fig. 5, B3 and C3). The cell nuclei that contain high levels of RPA foci show a nearly exact correlation to the nuclei that stain positive for BUdR (Fig. 5, B and C). As observed in Fig. 3, C-1027 induces RPA focus formation in virtually all cell nuclei, regardless of BUdR incorporation (Fig. 5D). These results suggest that adozelesin and CPT both trigger DNA damage-induced RPA foci only in S phase cells.
View larger version (35K):
[in this window]
[in a new window]
[Download PPT slide]
|
Fig 5. DNA damage-induced RPA foci in asynchronous cells labeled with BUdR. Asynchronously growing HeLa cells pulse labeled with BUdR were treated with the 50 µM CPT, 40 nM adozelesin, or 1 nM C-1027 for 1 h. The total DNA is visualized by DAPI stain (left panels). Cells labeled with BUdR (stained green) shown in the center panels represent cells in S phase. RPA stained with polyclonal antibody against RPA32 (in red) is shown in the right panels.
|
|
We then evaluated whether it is just S phase or actual replication fork progression that is required to induce RPA focalization. HeLa cells were enriched in S phase by incubating with aphidicolin for 16–18 h. As expected, these aphidicolin-arrested S phase cells (Fig. 6, left panels) showed no BUdR incorporation (data not shown). However, 1–2 h after their release from aphidicolin block, DNA replication activity resumed in these cells, and 100% of the cells showed BUdR incorporation (data not shown). We defined these cells as released S phase cells (Fig. 6, right panels). Aphidicolin-arrested or -released S phase cells were then treated with DNA-damaging agents for another 2 h and stained for extraction-resistant RPA as described above. Treatment with CPT or adozelesin did not appreciably alter RPA staining in the aphidicolin-arrested cells (Fig. 6, compare panels B2 and C2 with A2); however, they induced high levels of extraction-resistant RPA foci in virtually all cells on release from aphidicolin block (Fig. 6, B4 and C4). These results strongly suggest that CPT- and adozelesin-induced RPA foci are dependent not just on S phase but more specifically on replication fork progression. Treatment with C-1027 induced RPA focalization, regardless of S phase status or replication fork progression (Figs. 3D and 6D). It is noteworthy that although overnight aphidicolin treatment alone slightly enhanced RPA staining (compare Fig. 6A with Fig. 3A), even prolonged aphidicolin treatment did not induce appreciable RPA focalization.
View larger version (40K):
[in this window]
[in a new window]
[Download PPT slide]
|
Fig 6. Formation of DNA damage-induced RPA foci in S phase cells. HeLa cells were arrested in S phase by treating with 25 µM aphidicolin overnight. The cells were then incubated with 50 µM CPT, 40 nM adozelesin, or 1 nM C-1027 for 2 h either directly (left panels; Aphidicolin Arrested) or after cells were released from aphidicolin block for 1 h (right panels; Released). Cells were fixed and stained for DNA (with DAPI) and RPA (with monoclonal antibody against RPA32) as described in "Materials and Methods."
|
|
DNA Damage-induced RPA32 Hyperphosphorylation in S Phase Cells.
DNA damage-induced RPA32 hyperphosphorylation was also evaluated in aphidicolin-blocked or -released S phase cells. HeLa cells treated overnight with aphidicolin showed low levels of RPA32 phosphorylated to an intermediate mobility that were not appreciably changed after release from aphidicolin block (Fig. 7, Lanes 1 and 2). This likely represents the phosphorylation of RPA32 seen during S phase (6, 7). In several experiments, aphidicolin-arrested cells treated with either adozelesin or CPT showed either undetectable or very low levels of hyperphosphorylated RPA32 (Fig. 7, Lanes 3, 5, and 1 and data not shown). However, on release from aphidicolin block, which allows resumption of replication fork progression, >50% of the RPA32 in these cells became hyperphosphorylated (Fig. 7, Lanes 4 and 6). As with RPA focalization, C-1027 induced high levels of RPA32 hyperphosphorylation, regardless of S phase arrest or replication fork progression (Fig. 7, Lanes 7 and 8). These hyperphosphorylation results closely parallel those seen with RPA focalization (Fig. 6). Treatment with lower levels of these agents (as low as 1 nM adozelesin, 1 µM CPT, or 0.1 nM C-1027) resulted in decreased levels or intensity of -H2AX and RPA focalization and RPA32 hyperphosphorylation but showed the same cell cycle and replication fork dependence as seen with the higher levels of drugs, shown in the figures above (Refs. 3, 9, and 25 and data not shown).
View larger version (16K):
[in this window]
[in a new window]
[Download PPT slide]
|
Fig 7. DNA damage-induced RPA32 hyperphosphorylation in S phase cells. CPT (50 µM), 40 nM adozelesin, or 1 nM C-1027 were used to treat HeLa cells arrested with 25 µM aphidicolin for 2 h either directly (even numbered lanes) or after 1-h release from aphidicolin block (odd numbered lanes), as described in Fig. 6. Cells were lysed under reducing/denaturing conditions, and the lysates were separated by SDS-PAGE and immunoblotted for RPA32. Migration of RPA32 and hyperphosphorylated RPA32 are indicated on the right.
|
|
|
Discussion
|
|---|
On the basis of the results presented above, in conjunction with reports published previously, we propose a model for how various types of DNA damage are affected by replication fork progression (Fig. 8). It is clear that for genotoxic agents that directly cleave DNA, such as C-1027, the broken DNA ends are capable of inducing various cellular DNA damage responses (Fig. 8A). Replication fork movement is therefore not required for the induction of either RPA or -H2AX focus formation or for the activation of the RPA32 kinase. These results show that DNA damage-induced RPA focus formation and hyperphosphorylation can happen outside S phase of the cell cycle, in contrast to the model suggested by others (8–10). Gamma radiation also creates DSBs; however, RPA32 hyperphosphorylation induced by radiation was largely blocked by treatment with aphidicolin (8). This may be explained in that DSBs only make up 20% of the total DNA lesions in -irradiated cells (28). Some of these other lesions likely require DNA replication fork movement to induce cellular S phase checkpoint responses.
For indirect damage on DNA, such as CPT, or damage that is poorly recognized by cellular DNA repair pathways, such as DNA alkylation by adozelesin, the induction of RPA and -H2AX focalization and RPA32 hyperphosphorylation occurs only in S phase cells with active replication fork progression. CPT and its derivatives are among the most frequently used anticancer drugs and have long been known to selectively induce S phase DNA damage checkpoints (for review, see Ref. 29). Specifically, CPT treatment can induce DSBs in treated cells only if DNA replication forks are in motion (Fig. 8B; Ref. 12). This likely explains the S phase specificity of these drugs. In this study, we show for the first time that like RPA32 hyperphosphorylation, CPT-induced RPA and -H2AX focalization also require replication fork progression.
In contrast to CPT, the alkylation of DNA by adozelesin in vivo apparently does not cause an appreciable number of DNA strand breaks (30) or distort the duplex structure of targeted DNA (2). There have been some indications that CPI-induced lesions may be removed by nucleotide excision repair (31, 32). However, adducts on DNA caused by the CPI drug CC-1065 were found to persist in CC-1065-treated BSC-1 green monkey cells (33). The absence of RPA and -H2AX foci in non-S phase cells suggests that adozelesin-induced DNA adducts are either virtually undetectable or intractable to DNA repair enzymes, until the collision of replication forks with the drug:DNA adducts results in stalled replication forks. Stalled replication forks have been shown to induce S phase checkpoint responses (34, 35). Because adozelesin treatment does not induce apparent DNA strand breaks (30, 33),4 we conclude that adozelesin-induced, S phase-specific DNA damage and checkpoint responses must be triggered by stalled replication forks rather than DNA strand breaks. The S phase and replication dependence of DNA damage responses triggered by adozelesin are of particular relevance because adozelesin treatment results in cells arrested predominantly in S phase (3, 4).
Phosphorylation on serine 139 of histone H2AX by ATM is an early cellular response to DSBs. Both C-1027 and CPT are capable of inducing DSBs in treated cells; therefore, -H2AX focus formation is expected. However, adozelesin-induced -H2AX foci would have to result from a different mechanism. A recent publication showed that -H2AX foci are formed in response to replicational stress induced by either hydroxyurea or UV treatment (36). The kinase responsible for this phosphorylation was shown to be ATM-Rad3-related protein kinase rather than ATM. Because adozelesin-induced -H2AX focus formation is dependent on replication fork progression, this pathway may also be dependent on ATM-Rad3-related protein kinase. Although aphidicolin is capable of inhibiting DNA polymerase activity and blocking replication fork progression, RPA and -H2AX focus formation and RPA32 hyperphosphorylation were not triggered to an appreciable extent by aphidicolin treatment alone (Figs. 3, 6, and 7). Hence, replicational stress alone is not sufficient to induce RPA or -H2AX foci. This may indicate that there is more than one type of checkpoint response to stalled replication forks, depending on the manner of blockage.
|
Acknowledgments
|
|---|
We thank J. Newman for preparing the MBP-RPA32 antiserum, Dr. Michael O’Donnell for the His-RPA32 expression vector, and Dr. Xin Lin and Y. Ming Loo for critical reading of this manuscript. We also thank Drs. Joel Huberman and Ron Berezney for evaluation of an early draft of this manuscript and Dr. Marc Wold for insightful suggestions.
|
Footnotes
|
|---|
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.
1 Supported by NIH Grant CA 89259 (to T. M.). T. M. was also supported by an NIH Independent Scientist Award, K02 AI01686. S-R. K. was supported by a United States Army Breast Cancer Research Program Postdoctoral Traineeship, DAMD17-00-1-0418. T. A. B. was supported by NIH Grants CA 77491 and CA 16056. 
2 J-S. L. and S-R. K. contributed equally to this manuscript.
3 The abbreviations used are: CPI, cyclopropylpyrroloindole; RPA, replication protein A; CPT, camptothecin; DSB, double-strand break; DAPI, 4,6-diamidino-2-phenylindole; BUdR, bromo-uridine deoxyribonucleic acid; ATM, ataxia telangiectasia-mutated kinase.
4 T. A. Beerman, unpublished data.
Received 5/13/02;
revised 7/31/02;
accepted 11/19/02.
|
References
|
|---|
Yoon, J. H., and Lee, C. S. Sequence selectivity of DNA alkylation by adozelesin and carzelesin. Arch. Pharm. Res., 21:385
–390,1998
.[Medline]
Cameron, L., and Thompson, A. S. Determination of the structural role of the linking moieties in the DNA binding of adozelesin. Biochemistry, 39:5004
–5012,2000
.[CrossRef][Medline]
Wang, Y., Beerman, T. A., and Kowalski, D. Antitumor drug adozelesin differentially affects active and silent origins of DNA replication in yeast checkpoint kinase mutants. Cancer Res., 61:3787
–3794,2001
.[Abstract/Free Full Text]
Bhuyan, B. K., Smith, K. S., Adams, E. G., Wallace, T. L., Von Hoff, D. D., and Li, L. H. Adozelesin, a potent new alkylating agent: cell-killing kinetics and cell-cycle effects. Cancer Chemother. Pharmacol., 30:348
–354,1992
.[CrossRef][Medline]
Liu, J. S., Kuo, S. R., McHugh, M. M., Beerman, T. A., and Melendy, T. Adozelesin triggers DNA damage response pathways and arrests SV40 DNA replication through replication protein A inactivation. J. Biol. Chem., 275:1391
–1397,2000
.[Abstract/Free Full Text]
Wold, M. S. Replication protein A: a heterotrimeric, single-stranded DNA-binding protein required for eukaryotic DNA metabolism. Ann. Rev. Biochem., 66:61
–92,1997
.[CrossRef][Medline]
Iftode, C., Daniely, Y., and Borowiec, J. A. Replication protein A (RPA): the eukaryotic SSB. Crit. Rev. Biochem. Mol. Biol., 34:141
–180,1999
.[CrossRef][Medline]
Shao, R. G., Cao, C. X., Zhang, H., Kohn, K. W., Wold, M. S., and Pommier, Y. Replication-mediated DNA damage by camptothecin induces phosphorylation of RPA by DNA-dependent protein kinase and dissociates RPA:DNA-PK complexes. EMBO J., 18:1397
–1406,1999
.[CrossRef][Medline]
Rodrigo, G., Roumagnac, S., Wold, M. S., Salles, B., and Calsou, P. DNA replication but not nucleotide excision repair is required for UVC-induced replication protein A phosphorylation in mammalian cells. Mol. Cell. Biol., 20:2696
–2705,2000
.[Abstract/Free Full Text]
Oakley, G. G., Loberg, L. I., Yao, J., Risinger, M. A., Yunker, R. L., Zernik-Kobak, M., Khanna, K. K., Lavin, M. F., Carty, M. P., and Dixon, K. UV-induced hyperphosphorylation of replication protein a depends on DNA replication and expression of ATM protein. Mol. Biol. Cell, 12:1199
–1213,2001
.[Abstract/Free Full Text]
McHugh, M. M., Yin, X., Kuo, S. R., Liu, J. S., Melendy, T., and Beerman, T. A. The cellular response to DNA damage induced by the enediynes C-1027 and neocarzinostatin includes hyperphosphorylation and increased nuclear retention of Replication Protein A (RPA) and trans inhibition of DNA replication. Biochemistry, 40:4792
–4799,2001
.[CrossRef][Medline]
Strumberg, D., Pilon, A. A., Smith, M., Hickey, R., Malkas, L., and Pommier, Y. 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., 20:3977
–3987,2000
.[Abstract/Free Full Text]
Xu, Y. J., Zhen, Y. S., and Goldberg, I. H. C1027 chromophore, a potent new enediyne antitumor antibiotic, induces sequence-specific double-strand DNA cleavage. Biochemistry, 33:5947
–5954,1994
.[CrossRef][Medline]
Kirk, C. A., Goodisman, J., Beerman, T. A., Gawron, L. S., and Dabrowiak, J. C. Kinetics of cleavage of intra- and extracellular simian virus 40 DNA with the enediyne anticancer drug C-1027. Biophys. Chem., 63:201
–209,1997
.[CrossRef][Medline]
Brenot-Bosc, F., Gupta, S., Margolis, R. L., and Fotedar, R. Changes in the subcellular localization of replication initiation proteins and cell cycle proteins during G1- to S-phase transition in mammalian cells. Chromosoma, 103:517
–527,1995
.[Medline]
Krude, T. Chromatin assembly factor 1 (CAF-1) colocalizes with replication foci in HeLa cell nuclei. Exp. Cell Res., 220:304
–311,1995
.[CrossRef][Medline]
Dimitrova, D. S., Todorov, I. T., Melendy, T., and Gilbert, D. M. Mcm2, but not RPA, is a component of the mammalian early G1-phase prereplication complex. J. Cell Biol., 146:709
–722,1999
.[Abstract/Free Full Text]
Dimitrova, D. S., and Gilbert, D. M. Stability and nuclear distribution of mammalian replication protein A heterotrimeric complex. Exp. Cell Res., 254:321
–237,2000
.[CrossRef][Medline]
Gasior, S. L., Wong, A. K., Kora, Y., Shinohara, A., and Bishop, D. K. Rad52 associates with RPA and functions with rad55 and rad57 to assemble meiotic recombination complexes. Genes Dev., 12:2208
–2221,1998
.[Abstract/Free Full Text]
Golub, E. I., Gupta, R. C., Haaf, T., Wold, M. S., and Radding, C. M. Interaction of human rad51 recombination protein with single-stranded DNA binding protein. RPA. Nucleic Acids Res., 26:5388
–5393,1998
.[Abstract/Free Full Text]
Raderschall, E., Golub, E. I., and Haaf, T. Nuclear foci of mammalian recombination proteins are located at single-stranded DNA regions formed after DNA damage. Proc. Natl. Acad. Sci. USA, 96:1921
–1926,1999
.[Abstract/Free Full Text]
Choudhary, S. K., and Li, R. BRCA1 modulates ionizing radiation-induced nuclear focus formation by the replication protein A p34 subunit. J. Cell. Biochem., 84:666
–674,2002
.[CrossRef][Medline]
Sakamoto, S., Nishikawa, K., Heo, S. J., Goto, M., Furuichi, Y., Shimamoto, A., Chardon, P., and Tonjes, R. R. Werner helicase relocates into nuclear foci in response to DNA damaging agents and co-localizes with RPA and Rad51. Characterization of chromosomally assigned replication-competent gamma porcine endogenous retroviruses derived from a large white pig and expression in human cells. Genes Cells, 6:421
–430,2001
.[Abstract]
Redon, C., Pilch, D., Rogakou, E. P., Sedelnikova, O., Newrock, K., and Bonner, W. M. Histone H2A variants H2AX and H2AZ. Curr. Opin. Genet. Dev., 12:162
–169,2002
.[CrossRef][Medline]
Din, S., Brill, S. J., Fairman, M. P., and Stillman, B. Cell-cycle-regulated phosphorylation of DNA replication factor A from human and yeast cells. Genes Dev., 4:968
–977,1990
.[Abstract/Free Full Text]
Han, Y., Loo, Y. M., Militello, K. T., and Melendy, T. Interactions of the papovavirus DNA replication initiator proteins, bovine papillomavirus type 1 E1 and simian virus 40 large T antigen, with human replication protein A. J. Virol., 73:4899
–4907,1999
.[Abstract/Free Full Text]
Liu, J. S., Kuo, S. R., Yin, X., Beerman, T. A., and Melendy, T. DNA damage by the enediyne C-1027 results in the inhibition of DNA replication by loss of replication protein A function and activation of DNA-dependent protein kinase. Biochemistry, 40:14661
–14668,2001
.[CrossRef][Medline]
Sutherland, B. M., Bennett, P. V., Sidorkina, O., and Laval, J. Clustered DNA damages induced in isolated DNA and in human cells by low doses of ionizing radiation. Proc. Natl. Acad. Sci. USA, 97:103
–108,2000
.[Abstract/Free Full Text]
Liu, L. F., Desai, S. D., Li, T. K., Mao, Y., Sun, M., and Sim, S. P. Mechanism of action of camptothecin. Ann. N. Y. Acad. Sci., 922:1
–10,2000
.[CrossRef][Medline]
Cobuzzi, R. J., Jr., Burhans, W. C., and Beerman, T. A. Inhibition of initiation of simian virus 40 DNA replication in infected BSC-1 cells by the DNA alkylating drug adozelesin. J. Biol. Chem., 271:19852
–19859,1996
.[Abstract/Free Full Text]
Jin, S. G., Choi, J. H., Ahn, B., O’Connor, T. R., Mar, W., and Lee, C. S. Excision repair of adozelesin-N3 adenine adduct by 3-methyladenine-DNA glycosylases and UvrABC nuclease. Mol. Cell, 11:41
–47,2001
.
Gunz, D., Hess, M. T., and Naegeli, H. Recognition of DNA adducts by human nucleotide excision repair. Evidence for a thermodynamic probing mechanism. J. Biol. Chem., 271:25089
–25098,1996
.[Abstract/Free Full Text]
Zsido, T. J., Woynarowski, J. M., Baker, R. M., Gawron, L. S., and Beerman, T. A. Induction of heat-labile sites in DNA of mammalian cells by the antitumor alkylating drug CC-1065. Biochemistry, 30:3733
–3738,1991
.[CrossRef][Medline]
Rothstein, R., Michel, B., and Gangloff, S. Replication fork pausing and recombination or "gimme a break." Genes Dev., 14:1
–10,2000
.[Free Full Text]
Gottifredi, V., Shieh, S., Taya, Y., and Prives, C. From the Cover: p53 accumulates but is functionally impaired when DNA synthesis is blocked. Proc. Natl. Acad. Sci. USA, 98:1036
–1041,2001
.[Abstract/Free Full Text]
Ward, I. M., and Chen, J. Histone H2AX is phosphorylated in an ATR-dependent manner in response to replicational stress. J. Biol. Chem., 276:47759
–47762,2001
.[Abstract/Free Full Text]
This article has been cited by other articles:
|
|
|
|
 
D. R. Kennedy, L. S. Gawron, J. Ju, W. Liu, B. Shen, and T. A. Beerman
Single Chemical Modifications of the C-1027 Enediyne Core, a Radiomimetic Antitumor Drug, Affect Both Drug Potency and the Role of Ataxia-Telangiectasia Mutated in Cellular Responses to DNA Double-Strand Breaks
Cancer Res.,
January 15, 2007;
67(2):
773 - 781.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
M. M. McHugh, L. S. Gawron, S.-I. Matsui, and T. A. Beerman
The Antitumor Enediyne C-1027 Alters Cell Cycle Progression and Induces Chromosomal Aberrations and Telomere Dysfunction
Cancer Res.,
June 15, 2005;
65(12):
5344 - 5351.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
P. L. Olive, J. P. Banath, and L. T. Sinnott
Phosphorylated Histone H2AX in Spheroids, Tumors, and Tissues of Mice Exposed to Etoposide and 3-Amino-1,2,4-Benzotriazine-1,3-Dioxide
Cancer Res.,
August 1, 2004;
64(15):
5363 - 5369.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
W. D. Block, Y. Yu, and S. P. Lees-Miller
Phosphatidyl inositol 3-kinase-like serine/threonine protein kinases (PIKKs) are required for DNA damage-induced phosphorylation of the 32 kDa subunit of replication protein A at threonine 21
Nucleic Acids Res.,
February 10, 2004;
32(3):
997 - 1005.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
J. P. Banath and P. L. Olive
Expression of Phosphorylated Histone H2AX as a Surrogate of Cell Killing by Drugs That Create DNA Double-Strand Breaks
Cancer Res.,
August 1, 2003;
63(15):
4347 - 4350.
[Abstract]
[Full Text]
[PDF]
|
|
|
Top
Abstract
Introduction
