Cell Cycle Differences in DNA Damage-induced BRCA1 Phosphorylation Affect Its Subcellular Localization*

Shinya Okada and Toru OuchiDagger

From the Derald H. Ruttenberg Cancer Center, The Mount Sinai School of Medicine, New York University, New York, New York 10029

Received for publication, August 23, 2002, and in revised form, October 1, 2002

    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Phosphorylation of BRCA1 tumor suppressor protein is regulated during the cell cycle and in response to DNA damage. Several Ser/Thr kinases have been implicated in BRCA1 phosphorylation, including ATM/ATR, cdk2, and hChk2 kinases. In this study, phospho-Ser-specific antibodies recognizing Ser-988, -1423, -1497, and -1524 residues of BRCA1 were employed to study BRCA1 phosphorylation during the S and G2/M phases under conditions of DNA damage. We observed that IR (ionizing radiation) treatment induced phosphorylation of Ser-988/Ser-1524 during the S phase and of Ser-988/Ser-1423 during the G2/M phase. UV treatment induced phosphorylation of Ser-988 during the S phase and of Ser-1423 during the G2/M phase. Phosphorylation of serines 1423 and -1524 was not induced in HCC1937 breast cancer cells, which contain mutant BRCA1 protein. Confocal microscopy revealed that unphosphorylated BRCA1 localizes on chromosomes from metaphase through telophase, whereas Ser-988-phosphorylated BRCA1 resides in the inner chromosomal structure, centrosome, and the cleavage furrow during prophase through telophase. We also found that Ser-988-phosphorylated BRCA1 relocalizes to the perinuclear region when cells are subjected to IR or UV radiation in the S phase. These results reinforce a model wherein phosphorylation of specific residues of BRCA1 after DNA damage affects its localization and function.

    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Breast cancer tumor suppressor protein BRCA1 is a nuclear phosphoprotein with 1863 amino acids (1) that is implicated in the DNA repair pathway and regulation of gene transcription (2-7). In normally growing cells, BRCA1 is phosphorylated in a cell cycle-dependent manner; the protein undergoes hyperphosphorylation during the S phase and is dephosphorylated after the M phase (8, 9). Biochemical analysis has revealed that Ser-1497 is phosphorylated by cyclin-dependent kinase 2/cyclin A and E complexes (10). It has been also shown that DNA damage induces both nuclear redistribution of BRCA1 and an increased phosphorylation of the protein through DNA damage-activated kinases such as ATM,1 ATR, and hCds1/Chk2 (11-14). Several phosphorylation sites have been identified under these conditions, including Ser-988, -1423, -1387, and -1524. It has recently been shown that re-expression of wild type BRCA1 confers weak resistance to DNA damage-induced cell death in HCC1937 BRCA1 mutant breast cancer cell lines (12, 13, 15), whereas phosphorylation-defective BRCA1 alleles carrying Ser to Ala substitution of these residues did not rescue them from apoptosis. Furthermore, ionizing radiation (IR) treatment has been shown to induce phosphorylation of transcriptional corepressor CtIP, consequently releasing the protein from the BRCA1-containing complex leading to activation of transcriptional regulation of BRCA1 (16). These results provide a model of how DNA damage-induced phosphorylation of BRCA1 leads to transmission of signals that regulate gene expression. Previous studies have revealed that BRCA1 is involved in the G2-M checkpoint (17-19). Mouse embryonic fibroblasts carrying a targeted deletion of exon 11 of the BRCA1 gene showed a defective G2-M checkpoint accompanying unequal chromosome segregation, abnormal nuclear division, and aneuploidy. Interestingly, these phenotypes were also induced by overexpression of centrosome-associated Aurora-A/BTAK/STK15 kinase, which is frequently amplified in breast cancer cells (20-22). More recently, BRCA1 has been shown to localize in the mitotic centrosome, interacting with gamma -tublin (23, 24). Although these studies strongly suggest pivotal functions of BRCA1 in mitosis, physiological roles and regulation of BRCA1 phosphorylation in mitosis still require clarification.

In the current study, we investigated how BRCA1 phosphorylation is regulated during the cell cycle and in response to DNA damage. For these purposes, phospho-Ser-specific antibodies recognizing Ser-988, -1423, -1497, and -1524 residues of BRCA1 were generated and employed to study BRCA1 phosphorylation in the S and G2/M phases of cells with or without DNA damage. These results reinforce a model wherein phosphorylation of specific residues of BRCA1 after DNA damage affects its localization and function.

    EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Generation of Phospho-specific Antibodies and Monoclonal Antibodies for BRCA1-- Rabbit polyclonal phospho-Ser-specific antibodies for BRCA1 were generated by Research Genetics, Inc. against KLH-conjugated synthetic peptides as follows: S988,LFPIKSpFVKTKCK; S1423, EPGVERSSpPSKCPS; S1497, VLEQHGSpQPSNSY; S1524, QNRNYPSpQEELIK. Monoclonal antibody 21A8 was generated by immunizing mice with GST-BRCA1 (amino acids 1314-1863) in the Mount Sinai School of Medicine Core.

Western Blotting of BRCA1-- Cell extracts were prepared in EBC buffer (50 mM HEPES, pH 7.6, 250 mM NaCl, 0.1% Nonidet P-40, 5 mM EDTA, pH 8.0) with mixed protease inhibitor (Sigma). To detect changes in the mobility of p220-p240BRCA1, prolonged 5% SDS-PAGE was used in 25 mM Tris, 192 mM glycine, 0.1% SDS. After transferring, filters were blocked with 8% nonfat dried milk in TBS-T (20 mM Tris, pH 8, 0.9% NaCl, 0.5% Tween). The primary antibodies 21A8, Ab-1 (Calbiochem) and 8F7 (GeneTex) were used for 1 h at room temperature. Antibodies S988, S1423, S1497, and S1524 were used at 10 µg/ml. The secondary antibodies (Jackson ImmunoResearch) were peroxidase-conjugated anti-mouse IgG (H+L) or anti-rabbit IgG (H+L). Films were developed by ECL.

Cell Culture and Preparation of Total Cell Extracts-- HEK293, MCF7, and HCC1937 cells were obtained from ATCC. Cells were cultured in Dulbecco's modified Eagle's medium, 10% fetal bovine serum for HEK293 and MCF7 cells, or RPMI1640, 10% fetal bovine serum for HCC1937 cells. Cell extracts were prepared in EBC buffer (50 mM Tris, pH 8, 120 mM NaCl, 0.5% Nonidet P-40 (NP-40)), with the addition of 50 mM NaF, 1 mM sodium orthovanadate, 100 µg/ml polymethylsulfonyl fluoride, 20 µg/ml aprotinin, and 10 µg/ml leupeptin. IR was administered using MARK2 IRRADIATOR (J. L. Shepherd & Associates, San Fernando, CA).

Cell Fractionation-- Cells were washed with ice-cold phosphate-buffered saline and lysed by Lysis buffer (20 mM Hepes, pH 7.5, 20% glycerol, 10 mM NaCl, 1.5 mM MgCl2, 0.2 mM EDTA, 0.1% Triton X-100, 1 mM dithiothreitol, 1 mM phenylmethylsulfonyl fluoride, 50 µM leupeptin, and 50 µg/ml aprotinin). After centrifuge at 2,300 rpm at 4 °C, pellet was resuspended in nuclear extract buffer (Lysis buffer containing 500 mM NaCl). After rocking at 4 °C for 1 h, samples were centrifuged. Twenty µg of both nuclear and cytoplasmic samples were immunoblotted by 8F7 or nucleophosmin antibody (Cell Signaling Technology).

Flow Cytometry-- Cell cycles were synchronized by following a previously described protocol (26) and analyzed by FACSCalibur (BD PharMingen).

Immunostaining Analysis-- Cells were fixed for 1 h in phosphate-buffered saline, 3% paraformaldehyde, 2% sucrose solution, followed by 5 min of permeabilization at room temperature in Triton buffer (0.5% Triton X-100, 20 mM HEPES, 50 mM NaCl, 3 mM MgCl2, 300 mM sucrose). Blocking was done with 5% normal horse serum, 5% normal goat serum. BRCA1 was visualized using mouse monoclonal antibody 21A8, and Ser-988 phosphorylation was visualized using the polyclonal S988 antibody. All secondary antibodies used were species-specific antibodies from Jackson ImmunoResearch (Texas Red-X for mouse IgG and fluorescein isothiocyanate for rabbit IgG), used at 1:100 throughout. Nuclei were stained with DAPI. All images were collected with a Leica TCS-SP confocal laser scanning microscope and processed using Adobe Photoshop software.

    RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Differential Phosphorylation of BRCA1 in MCF7 Cells and BRCA1-mutated HCC1937 Cells-- Phospho-Ser-specific antibodies were raised and affinity purified against synthetic peptides containing phosphorylated Ser-988 (S988), -1423 (S1423), -1497 (S1497), and -1524 (S1524) of the BRCA1 protein (see "Experimental Procedures"). Specificity of these phospho-specific BRCA1 antibodies was examined by immunoblot analysis using cell lysates prepared from exponentially growing MCF7 cells with or without lambda -phosphatase treatment (Fig. 1A, left). These results demonstrated that S988, S1423, S1497, and S1524 antibodies recognize only phosphorylated forms of BRCA1. Affinity-purified mouse monoclonal antibody 21A8 was generated by immunizing mice with amino acids 1314-1863 of the BRCA1 protein. MCF7 cells were cell cycle synchronized, and BRCA1 phosphorylation was studied using the antibodies described above. Increased expression of the beta -form (about 240 kDa) of BRCA1 was detected in the S and G2/M phases by 21A8, presumably as a result of the hyperphosphorylation (Fig. 1A, right). Phosphorylation of Ser-988 and -1423 of alpha -form BRCA1 (about 220 kDa) weakly increased in the G2/M phase, but phosphorylation of Ser-1497 and -1524 residues did not change significantly during the cell cycle. Notably, despite high levels of the beta -form BRCA1 in the S and G2/M phases detected by 21A8, only S988 antibody weakly detected an increased signal of the beta -form, indicating that beta -form BRCA1 contains multiple phosphorylation sites other than Ser-988 in the S and G2/M phases.


View larger version (37K):
[in this window]
[in a new window]
 
Fig. 1.   Cell cycle-dependent and DNA damage-induced phosphorylation of BRCA1. A, specific recognition of phosphorylated BRCA1 by S988, S1423, S1497, and S1524 antibodies. Where indicated, cell lysates were treated with 400 units of lambda -phosphatase (left). Phosphorylation of Ser-988, -1423, -1497, and -1524 was studied by the indicated antibodies in synchronized MCF7 cells (right). Positions of two major products were shown as alpha  and beta . B and C, growing cell cultures of MCF7 and HCC1937 cells were treated with UV or IR of the indicated dosage. Cell lysates were prepared after 1 h, and BRCA1 protein level and its phosphorylation status were studied by the indicated antibodies.

Next, we compared the phosphorylation of wild type and mutant forms (5382insC) of BRCA1. Asynchronized MCF7 cells expressing wild type BRCA1 were treated with different dosages of UV (5, 10, 50, and 100 mJ/cm2) or IR (1, 5, 10, and 50 Gy), and BRCA1 phosphorylation was then determined (Fig. 1B). Like 21A8, anti-BRCA1 antibody 8F7 detected the alpha - and beta -forms of BRCA1 protein in UV- and IR-treated cells. Phosphorylation of Ser-988 was strongly induced by 1 Gy of IR or UV treatment (50-100 mJ/cm2) with slow migration at position beta . UV treatment also strongly induced Ser-1423 phosphorylation of the beta -form. Increased Ser-1524 phosphorylation was observed in the alpha -form of BRCA1 in response to 10-50 mJ/cm2 of UV or 10 Gy of IR treatment. These results suggest that DNA-damaged cells contain several species of BRCA1 of differing phosphorylation status.

The HCC1937 breast cancer cell line expresses 5382insC mutant BRCA1, resulting in formation of the immature BRCT domain (25). We analyzed how this mutant form of BRCA1 is phosphorylated after DNA damage (Fig. 1C). The mutant did not show a significant mobility shift after DNA damage. Unlike the case in MCF7 cells, both UV and IR induced Ser-988 phosphorylation and a slight mobility shift in a dose-dependent manner, whereas phosphorylation of Ser-1423 and -1524 was not induced to a significant degree by the same treatment. Significantly, HCC1937 cells re-expressing wild type BRCA1 have been found to be more resistant to IR damage than those expressing S988A mutant BRCA1 (13). These results suggest that C-terminal BRCA1 is required for phosphorylation of BRCA1 and that the functional C-terminal BRCT domain, which is lost in mutant BRCA1 in HCC1937 cells, plays a crucial role in survival after damage.

Phosphorylation of BRCA1 by IR in the S and G2/M Phases-- We next investigated how DNA damage-specific phosphorylation is regulated during the cell cycle. MCF7 cells were synchronized at the S or G2/M phase and treated with different doses of IR as indicated. Samples were collected 0.5, 1, 3, and 5 h after treatment, and BRCA1 phosphorylation was determined by means of S988, S1423, and S1524 antibodies. In the S phase (Fig. 2A), 21A8 consistently detected the alpha - and beta -forms of BRCA1. Ser-988 phosphorylation weakly increased in the alpha -form, but the beta -form was predominantly and strongly phosphorylated in response to higher doses of treatment. Ser-1423 phosphorylation weakly increased in response to all doses tested. Ser-1524 phosphorylation was strongly induced by 5 Gy or higher doses and sustained at least 5 h after treatment. Phosphorylation of BRCA1 in the G2/M phase was also studied (Fig. 2B). Antibody 21A8 detected both the alpha - and the beta -forms of BRCA1 throughout the time course. After damage, Ser-988 phosphorylation slightly increased in the alpha '-form detected between the alpha - and beta -forms. Ser-1423 was phosphorylated in the alpha -form as early as 3 h by 1 Gy of radiation, but 5 Gy or higher doses induced Ser-423 phosphorylation within 30 min. Ser-1524 phosphorylation did not increase significantly in the alpha -form, and 10 Gy or higher doses of radiation even decreased phosphorylation.


View larger version (48K):
[in this window]
[in a new window]
 
Fig. 2.   IR-induced BRCA1 phosphorylation in S and G2/M phase. MCF7 cells were cell cycle synchronized at S (A) or G2/M (B) phase and treated with different dosages of IR. Samples were prepared after 0.5, 1, 3, and 5 h, and the phosphorylation status was studied by the indicated antibodies. The molecular weight of the alpha - and beta -forms corresponds to those in Fig 1.

These results demonstrate that (i) phosphorylation sites of BRCA1 are determined by both the level of DNA damage and the time course of unfolding of the effects of that damage, and (ii) after DNA damage BRCA1 exists as a heterogeneous pool of species with differing phosphorylation status even when the cell cycle has been synchronized.

Phosphorylation of BRCA1 by UV Radiation in the S and G2/M Phases-- We next studied UV-induced BRCA1 phosphorylation. Monoclonal antibody 21A8 predominantly detected the alpha -form of BRCA1 in damaged cells in the S phase (Fig. 3A). Reduced expression of BRCA1 in cells treated with high UV doses is presumably because of protein degradation of apoptotic cells. The BRCA1 alpha "-form, similar in molecular weight to the alpha '-form in Fig. 2B, showed Ser-988 phosphorylation when treated with 50 and 100 mJ/cm2 of UV after 3-5 h. Ser-1423 phosphorylation did not change in response to the lower dose but slightly decreased with 50 and 100 mJ/cm2 of UV. No significant changes of Ser-1524 phosphorylation were detected.


View larger version (37K):
[in this window]
[in a new window]
 
Fig. 3.   UV-induced BRCA1 phosphorylation in S and G2/M phase. MCF7 cells were cell cycle synchronized at S (A) or G2/M (B) phase and treated with different dosages of UV. Immunoblot analysis was done by the same procedures described in Fig 2. The alpha "-form represents the molecular weight between alpha - and beta -forms shown in Fig 1.

Antibody 21A8 was found to recognize hyperphosphorylated BRCA1 (beta -form) in the G2/M phase, although the antibody had been generated by immunizing a bacterial GST fusion protein that presumably is an unphosphorylated peptide (Fig. 3B). S988 and S1423 detected doublet signals (alpha - and beta -forms) when treated with 50 and 100 mJ/cm2 of UV. In the alpha -form Ser-1524 phosphorylation was slightly increased by 50 mJ/cm2 of UV. Again, these results demonstrate that several species of BRCA1 with differential phosphorylation status are present in UV-damaged S and G2/M phase cells.

Mitosis-specific Localization of Ser-988-phosphorylated BRCA1-- Cell cycle-dependent localization of BRCA1 was determined in detail by confocal laser microscopy using the 21A8 and S988 antibodies (Fig. 4A). As reported previously, nuclear BRCA1 was weakly stained during the G0 and G1 phases, and a distinct dot pattern appeared during S phase (26). A large nuclear dot pattern was induced 1 h after cells were treated with IR or UV but disappeared after 5 h. Although weak cytoplasmic dots were recognized by S988 during S phase, UV or IR damage induced a perinuclear dot pattern and nuclear signal. Redistribution of BRCA1 in DNA-damaged cells was confirmed by subcellular fractionation (Fig. 4B). Nuclear and cytoplasmic extracts of S phase cells were prepared at 1 h post-UV and -IR treatment. Although BRCA1 was predominantly nuclear in untreated S phase cells, DNA damage induced cytoplasmic redistribution of BRCA1.


View larger version (99K):
[in this window]
[in a new window]
 
Fig. 4.   Cell cycle-dependent and DNA damage-induced localization of Ser-988-phosphorylated BRCA1 in MCF7 cells. A, confocal microscopy was employed to determine the distribution of BRCA1 protein under conditions of DNA damage. S phase cells were treated by UV (10 mJ/cm2) or IR (5 Gy) and immunostained after 1 or 5 h. Arrows indicate the centrosome in metaphase and the cleavage furrow in cytokinesis, respectively. B, subcellular fractionation of BRCA1 in S phase cells with or without DNA damage (UV, 10 mJ/cm2; IR, 5 Gy). Samples were prepared 1 h after damage and immunoblotted by antibodies for BRCA1 (8F7) or nucleophosmin.

Although BRCA1 was visualized in both chromosome and cytoplasm during mitosis, S988 detected BRCA1 localized to the centrosome and microtubules only during metaphase; BRCA1 localization to the inner structure of the chromosome was also detected during anaphase and telophase. Interestingly, Ser-988-phosphorylated BRCA1 was also detected in the cleavage furrow during telophase and cytokinesis. These results suggest that specific phosphorylation of BRCA1 may be important for the regulation of mitosis.

    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

In the present studies, generation of S988, S1423, S1497, and S1524 antibodies enabled us to further investigate the regulation of BRCA1 phosphorylation in different conditions. Our results demonstrate that BRCA1 exists as a heterogeneous species of differentially phosphorylated status and that phosphorylation of each Ser residue examined is specifically induced in cell cycle and in response to DNA damage. Thus, at damaged state, BRCA1 exists as a pool of species with different phosphorylation status. It is likely that phosphorylation of specific residues determines a role and localization of BRCA1 in damaged cells.

Human Cds1 kinase (hCds1/Chk2) has been shown to colocalize BRCA1 and phosphorylate Ser-988 residue (12). In that study, low dosage of gamma -radiation (10 Gy) was sufficient to induce Ser-988 phosphorylation. Our results were consistent with these data in that Ser-988 phosphorylation was detected by 1 Gy of ionizing radiation in MCF7 cells. We also found that phosphorylation of BRCA1 Ser-988 is increased when IR-damaged in S phase (Fig. 2A), whereas it is not obvious in G2/M phase (Fig. 2B) although Chk2 kinase can respond to DNA damage throughout the cell cycle (27). It remains unclear whether hCds1/Chk2 colocalizes BRCA1 in G2/M phase. Significantly, HCC1937 cells re-expressing wild type BRCA1 have been found to be more resistant after gamma -radiation damage than those expressing S988A mutant BRCA1 (12). Our studies show that Ser-988 of truncated BRCA1 in HCC1937 cells is similarly phosphorylated with wild type BRCA1 in MCF7 cells. Taken together, these studies provide an implication that the phosphorylation of Ser-988 is not sufficient for damage-resistant phenotype and that the functional C-terminal BRCT domain, which is lost in mutant BRCA1 in HCC1937 cells, plays a crucial role for survival after damage.

Several other Ser residues have been identified as potential phosphorylation sites by ATM, including Ser-1387, -1423, -1457, and -1524 (13, 28, 29). Ser-1423 and -1524 are associated with the regulation of cell growth after IR; HCC1937 cells re-expressing wild type BRCA1 can grow after IR damage, whereas cells expressing a phosphorylation-deficient mutant of these Ser residues show growth retardation under the same condition (13, 15). It is tempting to speculate that the allosteric change of BRCA1 structure because of phosphorylation affects the interaction between BRCA1 and other proteins involved in the DNA damage response. Among them are BRCA2 and the Rad50-Mre11-Nbs complex, but it has been found that damage-dependent phosphorylation does not change the amount of BRCA1 associated with these complexes (30, 31).

We found that DNA damage induces both nuclear dot pattern and perinuclear distribution of Ser-988-phosphorylated BRCA1. Recently, it was reported that BRCA1 can shuttle between nucleus and cytoplasm and that a Rev-type nuclear export sequence near the N terminus facilitates export through the CRM exporting pathway (32). These results have also shown that DNA damage (actinomycin D treatment) resulted in the accumulation of BRCA1 in the cytoplasm, suggesting that DNA damage induces shuttling of the BRCA1 protein, although whether Ser-988 phosphorylation is necessary for the relocalization of the BRCA1 protein remains to be determined. Studies are in progress to elucidate whether Ser-988 can be phosphorylated when export is inhibited by Leptomycin B, a specific inhibitor of CRM-dependent BRCA1 nuclear export1.

Antibody 21A8 detected chromosome structure throughout mitosis, and Ser-988-phosphorylated BRCA1 was detected in both cytoplasm and the inner mitotic chromosome in prophase, anaphase, and telophase. In metaphase, centrosome and spindle structures were strongly stained by S988. Although we could not determine precise localization of Ser-988-phosphorylated BRCA1 in the inner chromosome, this localization may represent the mitosis-specific structure, such as the kinetochore. Supporting this hypothesis, BRCA2, which has been shown to colocalize with BRCA1 in mitosis (29), was shown to associate with hBUBR1, a component of the kinetochore CEMP-E/F complex (33).

In summary, the present results demonstrate that BRCA1 phosphorylation sites in response to DNA damage are determined by the cell cycle and the dosage of the damage and that BRCA1 exist as a heterogeneous pool of species with differential phosphorylation status even when cell cycle has been synchronized. Our results raised several issues that are potentially important for the tumor suppressive function of BRCA1 in the DNA damage pathway; biochemical details of signaling conferred by phosphorylated BRCA1 will be necessary to understand the role of this protein in this pathway.

    ACKNOWLEDGEMENTS

We thank Drs. David M. Livingston and Sam Lee at Harvard Medical School for discussion, Mutsuko Ouchi for generating and characterizing monoclonal anti-BRCA1 antibodies, and Dr. Scott Henderson for confocal laser microscope analysis at The Mount Sinai School of Medicine Microscopy Core Facilities.

    FOOTNOTES

* This work was supported by the New York City Council Speaker's Fund, an Empowerment through Innovative Research and Education (EMPIRE) grant by the State of New York, and NCI, National Institutes of Health Grants CA79892 and CA90631 (to T. O.).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Dagger To whom correspondence should be addressed: Derald H. Ruttenberg Cancer Center, The Mount Sinai School of Medicine, New York University, Box 1130, 1 Gustave L. Levy Pl., New York, NY 10029. Tel.: 212-659-5475; Fax: 212-987-2240; E-mail: Toru.Ouchi@mssm.edu.

Published, JBC Papers in Press, November 8, 2002, DOI 10.1074/jbc.M208685200

    ABBREVIATIONS

The abbreviations used are: ATM, ataxia telangiectasia mutated; ATR, ATM-related kinase; DAPI, 4',6-diamidino-2'-phenylindole; IR, ionizing radiation; Gy, gray.

    REFERENCES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

1. Miki, Y., Swensen, J., Shattuck-Eidens, D., Futreal, P. A., Harshman, K., et al.. (1994) Science 266, 66-71[Medline] [Order article via Infotrieve]
2. Somasundaram, K., Zhang, H., Zeng, Y.-X., Houvras, Y., Peng, Y., Zhang, H., Wu, G. S., Licht, J. D., Weber, B. L., and El-Deiry, W. S. (1997) Nature 389, 187-190[CrossRef][Medline] [Order article via Infotrieve]
3. Ouchi, T., Monteiro, A. N., August, A., Aaronson, S. A., and Hanafusa, H. (1998) Proc. Natl. Acad. Sci. U. S. A. 95, 2302-2306[Abstract/Free Full Text]
4. Ouchi, T., Lee, S. W., Ouchi, M., Aaronson, S. A., and Horvath, C. M. (2000) Proc. Natl. Acad. Sci. U. S. A. 97, 5208-5213[Abstract/Free Full Text]
5. Zhang, H., Somasundaram, K., Peng, Y., Tian, H., Zhang, H., Bi, D., Weber, B. L., and El-Deiry, W. S. (1998) Oncogene 16, 1713-1721[CrossRef][Medline] [Order article via Infotrieve]
6. Scully, R., and Livingston, D. M. (2000) Nature 408, 429-432[CrossRef][Medline] [Order article via Infotrieve]
7. Deng, C. X., and Brodie, S. G. (2000) Bioessays 8, 728-737[CrossRef]
8. Chen, Y., Farmer, A. A., Chen, C.-F., Jones, D. C., Chen, P.-L., and Lee, W.-H. (1996) Cancer Res. 56, 3168-3172[Abstract]
9. Ruffner, H., and Verma, I. M. (1997) Proc. Natl. Acad. Sci. U. S. A. 94, 7138-7143[Abstract/Free Full Text]
10. Ruffner, H., Jiang, W., Craig, A. G., Hunter, T., and Verma, I. M. (1999) Mol. Cell. Biol. 19, 4843-4854[Abstract/Free Full Text]
11. Chen, J. (2000) Cancer Res. 60, 5037-5039[Abstract/Free Full Text]
12. Lee, J. S., Collins, K. M., Brown, A. L., Lee, C. H., and Chung, J. H. (2000) Nature 404, 201-204[CrossRef][Medline] [Order article via Infotrieve]
13. Cortez, D., Wang, Y, Qin, J., and Elledge, S. J. (1999) Science 286, 1162-1166[Abstract/Free Full Text]
14. Tibbetts, R. S., Cortez, D., Brumbaugh, K. M., Scully, R., Livingston, D., Elledge, S. J., and Abraham, R. T. (2000) Genes Dev. 14, 2989-3002[Abstract/Free Full Text]
15. Scully, R., Ganesan, S., Vlasakova, K., Chen, J., Socolovsky, M., and Livingston, D. M. (1999) Mol. Cell 4, 1093-1099[Medline] [Order article via Infotrieve]
16. Li, S., Ting, N. S., Zheng, L., Chen, P.-L., Ziv, Y., Shiloh, Y., Lee, E.-Y., and Lee, W. H. (2000) Nature 406, 210-215[CrossRef][Medline] [Order article via Infotrieve]
17. Xu, X., Weaver, Z., Linke, S. P., Li, C., Gotay, J., Wang, X.-W., Harris, C. C., Ried, T., and Deng, C.-X. (1999) Mol. Cell. 3, 389-395[Medline] [Order article via Infotrieve]
18. Xu, B., Kim, S.-T., and Kastan, M. B. (2001) Mol. Cell. Biol. 21, 3445-3450[Abstract/Free Full Text]
19. Yarden, R., Pardo-Reoyo, S., Sgagias, M., Cowan, K. H., and Brody, L. C. (2002) Nat. Genet. 30, 285-289[CrossRef][Medline] [Order article via Infotrieve]
20. Sen, S., Zhou, H., and White, R. A. (1997) Oncogene 14, 2195-2200[CrossRef][Medline] [Order article via Infotrieve]
21. Zhou, H., Kuang, J., Zhong, L., Kuo, W.-L., Gray, J. W., Sahin, A., Brinkley, B. R., and Sen, S. (1998) Nat. Genet. 20, 189-193[CrossRef][Medline] [Order article via Infotrieve]
22. Tanner, M. M., Grenman, S., Koul, A., Johannsson, O., Meltzer, P., Pejovic, T., Borg, A., and Isola, J. J. (2000) Clin. Cancer Res. 6, 1833-1839[Abstract/Free Full Text]
23. Hsu, L.-C., and White, R. L. (1998) Proc. Natl. Acad. Sci. U. S. A. 95, 12983-12988[Abstract/Free Full Text]
24. Hsu, L.-C., Doan, T. P., and White, R. L. (2001) Cancer Res. 61, 7713-7718[Abstract/Free Full Text]
25. Tomlinson, G. E., Chen, T. T., Stastny, V. A., Virmani, A. K., Spillman, M. A., Tonk, V., Blum, J. L., Schneider, N. R., Wistuba, I. I., Shay, J. W., Minna, J. D., and Gazdar, A. F. (1998) Cancer Res. 58, 3237-3242[Abstract]
26. Scully, R., Chen, J., Ochs, R. L., Keegan, K., Hoekstra, M., Feunteun, J., and Livingston, D. M. (1997) Cell 90, 425-435[Medline] [Order article via Infotrieve]
27. Matsuoka, S., Huang, M., and Elledge, S. J. (1998) Science 282, 1893-1897[Abstract/Free Full Text]
28. Gatei, M., Scott, S. P., Filippovitch, I., Soronika, N., Lavin, M. F., Weber, B., and Khanna, K. K. (2000) Cancer Res. 60, 3299-3304[Abstract/Free Full Text]
29. Gatei, M., Zhou, B. B., Hobson, K., Scott, S., Young, D., and Khanna, K. K. (2001) J. Biol. Chem. 276, 17276-17280[Abstract/Free Full Text]
30. Chen, J., Silver, D. P., Walpita, D., Cantor, S. B., Gazdar, A. F., Thomlinson, G., Couch, F. J., Weber, B. L., Ashley, T., Livingston, D. M., and Scully, R. (1998) Mol. Cell. 2, 317-328[Medline] [Order article via Infotrieve]
31. Zhong, Q., Chen, C.-F., Li, S., Chen, Y., Wang, C. C., Xiao, J., Chen, P. L., Sharp, Z. D., and Lee, W. H. (1999) Science 285, 747-750[Abstract/Free Full Text]
32. Rodriguez, J. A., and Henderson, B. R. (2000) J. Biol. Chem. 275, 38589-38596[Abstract/Free Full Text]
33. Futamura, M., Arakawa, H., Matsuda, K., Katagiri, T., Saji, S., Miki, Y., and Nakamura, Y. (2000) Cancer Res. 60, 1531-1535[Abstract/Free Full Text]


Copyright © 2003 by The American Society for Biochemistry and Molecular Biology, Inc.