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Genes to Cells (2004) 9, 131-142. doi:10.1111/j.1356-9597.2004.00710.x
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Down-regulation of nuclear protein ICBP90 by p53/p21Cip1/WAF1-dependent DNA-damage checkpoint signals contributes to cell cycle arrest at G1/S transition

Yoshimi Arima1, Toru Hirota1, Christian Bronner2, Marc Mousli2, Toshiyoshi Fujiwara3, Shin-ichiro Niwa4, Hiroyuki Ishikawa4 and Hideyuki Saya1,*

1 Department of Tumour Genetics and Biology, Graduate School of Medical Sciences, Kumamoto University, Kumamoto 860-8556, Japan
2 Inserm UMR S392, Faculté de Pharmacie, 74 route du Rhin, BP 60024, 67401 Illkirch Cedex, France
3 Division of Surgical Oncology, Department of Surgery, Okayama University Graduate School of Medicine and Dentistry, Okayama 700-8558, Japan
4 Department of Biomedical Research & Development, Link Genomics, Tokyo 103-0023, Japan


   Abstract
Top
 Abstract
Introduction
 Results
 Discussion
 Experimental procedures
 Supplementary material
 References
 
Checkpoints, which monitor DNA damage and regulate cell cycle progression, ensure genomic integrity and prevent the propagation of transformed cells. DNA damage activates the p53-dependent checkpoint pathway that induces expression of p21Cip1/WAF1, resulting in cell cycle arrest at G1/S transition by inhibition of cdk activity and DNA replication. ICBP90 was identified as a nuclear protein that binds to the TopoII{alpha} gene promoter and is speculated to be involved in DNA replication. ICBP90 expression is cell cycle regulated in normal cells but stably high throughout cell cycle in various cancer cell lines. We here demonstrate that ICBP90 expression is down-regulated by the p53/p21Cip1/WAF1-dependent DNA damage checkpoint signals. The reduction of ICBP90 appeared to be caused by both transcriptional suppression and protein degradation. Adenoviral expression of p21Cip1/WAF1 directly led to ICBP90 reduction in p53–/– HCT116 cells without DNA damage. Furthermore, ICPB90 depletion by RNA interference significantly blocked G1/S transition after DNA damage in HeLa cells. The down-regulation of ICBP90 is an important mechanism for cell cycle arrest at G1/S transition, which is induced by the activation of a p53/p21Cip1/WAF1-dependent DNA-damage checkpoint. Deregulation of ICBP90 may impair the control of G1/S transition during checkpoint activation and lead to genomic instability.


   Introduction
Top
 Abstract
 Introduction
Results
 Discussion
 Experimental procedures
 Supplementary material
 References
 
Checkpoints are a regulatory mechanism that ensures genomic integrity and prevents the propagation of transformed cells (Hartwell & Kastan 1994). Checkpoints control a signalling system that changes the activity of cyclin-dependent kinases (cdk), delaying cell cycle progression and preventing transition into subsequent phases until all processes in the previous phase are completed (Hartwell & Weinert 1989). Arrest in G1 is considered to prevent aberrant replication of damaged DNA and arrest in G2 allows cells to avoid segregation of defective chromosomes. Thus, defective cell cycle checkpoints lead to gene mutations, chromosome damage, and aneuploidy, all of which contribute to tumorigenesis (Hartwell & Kastan 1994; Paulovich et al. 1997).

G1 arrest after DNA damage is induced primarily by stabilization of the p53 tumour suppressor protein (Lakin & Jackson 1999). p53 is activated and post-translationally modified in response to DNA damage. These modifications include phosphorylation by ataxia telangiectasia mutated (ATM) kinase, which is implicated in DNA damage-signalling pathways (Canman et al. 1998; Khanna et al. 1998). p53 can act as a transcription factor that, in response to cellular stress, binds DNA in a sequence-specific manner and induces the expression of genes containing a p53 binding-site element in their promoter or introns (Bourdon et al. 1997; el-Deiry et al. 1992; Funk et al. 1992). The cdk inhibitor p21Cip1/WAF1 was identified as a p53-responsive gene and plays a critical role in p53-induced G1 arrest. The cell cycle inhibitory activities of p21Cip1/WAF1 are attributable to not only inhibition of cdk activity but also several other processes, including interaction with proliferating cell nuclear antigen (PCNA) to inhibit DNA replication (Chen et al. 1995; Luo et al. 1995; Sherr & Roberts 1995; Waga et al. 1994).

ICBP90 (Inverted CCAAT box Binding Protein, 90 kD) was identified recently and is a nuclear protein that binds to one of the inverted CCAAT boxes (called ICB2) of the topoisomerase II{alpha} (TopoII{alpha}) gene promoter (Hopfner et al. 2000, 2002). ICBP90 contains an N-terminal ubiquitin-like domain partially overlapped by a leucine zipper domain, followed by a central zinc finger of the PHD finger type and a C-terminal zinc finger of the RING finger type. It also has two retinoblastoma protein (pRb)-binding sites in the PHD finger and the RING finger domains. Expression of ICBP90 is observed throughout cell cycle but peaks at late G1 and during G2/M phases in non-cancerous human cells. However, in various cancer cells, ICBP90 expression is much higher compared to normal cells and the cell-cycle dependent expression pattern is abolished (Mousli et al. 2003). Furthermore, the expression of ICBP90 is concomitant with that of TopoII{alpha} in normal cells and cancer cells, and transfection of ICBP90 into COS1 cells has resulted in enhanced expression of TopoII{alpha} (Hopfner et al. 2000, 2002). Given that TopoII{alpha} expression is heavily linked to cell growth and sensitivity to cancer treatment (reviewed in Isaacs et al. 1998; Nitiss et al. 1998), ICBP90 may participate in the regulation of cell proliferation and cell cycle progression.

A mouse nuclear phosphoprotein Np95 was found to have similar domain structures with 73.6% of identity to ICBP90 and, thereby, is potentially a mouse counterpart of ICBP90 (Fujimori et al. 1998). Np95 is an early target of adenovirus E1A, and the concomitant expression of Np95 and cyclin E/cdk2 is sufficient to S phase progression (Bonapace et al. 2002). Moreover, recent studies have revealed that functional ablation of Np95 results in hypersensitivity to replication blocks and DNA damaging agents (Muto et al. 2002). All these earlier observations raise the possibility that Np95/ICBP90 contributes to a DNA-damage induced checkpoint-signalling pathway.

In this study, we demonstrate that expression of ICBP90 is regulated by the p53-dependent DNA damage checkpoint signals. We found that ICBP90 was down-regulated when p53+/+ HCT116 cells were treated with adriamycin to induce DNA damage, but not in p53–/– HCT116 cells. This down-regulation of ICBP90 by DNA damage appeared to be caused by both the reduction of transcription and the promotion of a ubiquitin/proteasome-dependent proteolytic pathway. Furthermore, we found that expression of p21Cip1/WAF1 induced by DNA-damage-mediated p53 activation directly regulated the reduction of ICBP90. Our findings suggest that ICBP90 is one of the targets for p53/p21Cip1/WAF1-mediated checkpoint pathway and plays a role in cell cycle arrest at G1/S transition.


   Results
Top
 Abstract
 Introduction
 Results
Discussion
 Experimental procedures
 Supplementary material
 References
 
Down-regulation of ICBP90 expression by DNA damage in HCT116 cells

It has been shown that ICBP90 expression is much higher in various human cancer cell lines, such as HeLa, Jurkat and A549, compared to normal cells (Mousli et al. 2003). Immunocytochemical analysis showed that HCT116 colon cancer cells, which have normal p53 and a near-diploid karyotype and thus, in principle, closely resemble normal human cells (Lengauer et al. 1997), also expressed high levels of ICBP90; but that a small number of HCT116 cells were negative for ICBP90 staining (Fig. 1). This observation prompted us to investigate the types of intracellular signalling that can reduce the expression of ICBP90 in HCT116 cells. First, to analyse the cell cycle dependency of ICBP90 expression, the protein levels of ICBP90 were examined in HCT116 cells at different time points after release from nocodazole-induced metaphase block. Fluorescence-activated cell sorting (FACS) analysis showed that HCT116 cells were arrested at metaphase by nocodazole treatment and progress into G1 from M after release (Fig. 2A). Immunoblot analysis revealed that levels of ICPB90 expression in HCT116 cells did not change significantly throughout the cell cycle (Fig. 2B), which is consistent with data reported previously using other cancer cell lines (Mousli et al. 2003).



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  		Figure 1  Nuclear localization of the endogenous ICBP90 in HCT116 cells. (A, D) Distribution of endogenous ICBP90 in HCT116 cells was subjected to indirect immunofluorescence staining with 1RC1C-10, the anti-ICBP90 monoclonal antibodies (FITC, green). (B, E) DNA was visualized by staining with propidium iodide (PI, red). (C, F) The merged images are shown. Endogenous ICBP90 was detected in nuclei. Arrows indicate cells negative for ICBP90 expression (A–C). The percentage of ICBP90-negative cells cultured in normal conditions was 4.35%. In cells at the mitotic phase indicated by arrowheads, ICBP90 staining was detected only in the cytoplasmic area, not on chromosomes (D–F).

 


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  		Figure 2  ICBP90 expression is deregulated in HCT116 cells during the cell cycle. (A) HCT116 cells were synchronized at metaphase by nocodazole (100 ng/ml, 12 h) and released into fresh medium. The cells were harvested for FACS analysis after the indicated time, as described in ‘Experimental Procedures’. (B) Whole cell extracts harvested at the indicated times after nocodazole release were subjected to immunoblot analysis with antibodies to ICBP90 or cyclin B. Cyclin B levels were monitored for cell cycle phase of the harvested cells.

 
In the course of the immunocytochemical analysis of HCT116 cells cultured under normal conditions, we found that a small number of cells are stained with antibodies against phosphorylated histone H2AX, which is a marker for DNA double strand breaks, and those cells tend to be negative for ICBP90 staining (data not shown). Double-strand breaks in DNA induce the rapid phosphorylation of histone H2AX on serine-139 (Rogakou et al. 1998), which then results in the formation of {gamma}-H2AX complexes that can be detected by the antibodies (Rogakou et al. 1999). Therefore, we tested the possibility that DNA damage affects the levels of ICBP90 in HCT116 cells. Treatment with adriamycin for 36 h induced a marked increase in the number of {gamma}-H2AX-positive spots apparent in the nuclei and the concomitant reduction of ICBP90 expression (Fig. 3A). Immunoblot analysis also showed a significant down-regulation of ICBP90 levels with activation of p53/p21Cip1/WAF1 pathway by adriamycin treatment (Fig. 3B). The reduction of ICBP90 expression by adriamycin treatment was also observed in normal human fibroblasts, MRC-5 (supplemental figure S1).



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  		Figure 3  DNA double strand breaks induce ATM-dependent ICBP90 down-regulation in HCT116 cells. (A) Down-regulation of ICBP90 after adriamycin treatment. HCT116 cells were treated with adriamycin at a final concentration of 500 ng/ml to induce DNA double strand breaks. After 36 h adriamycin treatment, HCT116 cells were subjected to indirect immunofluorescence staining with the anti-ICBP90 (FITC, green, in upper panels) and phospho-H2AX (Texas Red, red, in middle panels) antibodies. The merged images are shown in bottom panels. (B) The protein levels of ICBP90 in HCT116 cells treated with adriamycin for 24 h. To inhibit ATM-dependent DNA damage signals, cells were pretreated with 5 mM caffeine for 1 h and then incubated with adriamycin (500 ng/ml). Whole cell extracts were subjected to immunoblot analysis with antibodies to ICBP90, p53, phospho-p53 (Ser15) or p21Cip1/WAF1. Note that adriamycin treatment down-regulated ICBP90 expression, whereas it promoted expression of p53 and p21Cip1/WAF1 and phosphorylation of Ser15 site of p53. Caffeine treatment reversed the effects of adriamycin on the expressions of those proteins and phosphorylation of Ser15 site of p53.

 
Serine-15 site of p53 is known to be phosphorylated by ATM kinase activated by double strand breaks of DNA (Banin et al. 1998). Therefore, to investigate the involvement of ATM-mediated DNA damage signals in ICBP90 reduction, we treated HCT116 cells with adriamycin in the presence of caffeine which is known to inhibit DNA-damage induced ATM kinase activation (Zhou et al. 2000). The reduction of ICBP90 by DNA damage was effectively blocked by treatment with caffeine (Fig. 3B), suggesting that ICBP90 levels are regulated by DNA damage-induced checkpoint signals.

ICBP90 is down-regulated by both transcriptional suppression and protein degradation after DNA damage

We next attempted to determine whether ICBP90 is down-regulated at the transcription or protein levels after DNA damage. Semiquantitative RT-PCR analysis revealed that mRNA expression levels of ICBP90 were markedly suppressed in adriamycin-treated HCT116 cells compared to untreated cells (Fig. 4A), suggesting that reduced transcription is a cause of ICBP90 down-regulation. However, treatment with {alpha}-amanitin, an inhibitor of RNA polymerase II-dependent transcription (Jacob et al. 1970; Lindell et al. 1970), also suppressed the expression of ICBP90 transcription (Fig. 4A) although it did not induce the down-regulation of ICBP90 protein in HCT116 cells (Fig. 4B). Therefore, we speculated that the suppression of transcription alone may not be sufficient to down-regulate ICBP90 and that protein degradation might be involved in this process. To test this possibility, we treated HCT116 cells with adriamycin in the presence of carbobenzoxy-L-leucyl-L-leucyl-L-leucinal (MG132), an inhibitor for various intracellular proteases as well as proteasome. The decline of ICBP90 levels in adriamycin-treated cells was effectively prevented by the presence of MG132 (Fig. 4B). These data indicate that down-regulation of ICBP90 is caused not only by reduced mRNA levels but also by proteolytic cleavage and/or ubiquitin/proteasome-mediated protein degradation under the activation of a DNA-damage checkpoint pathway.



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  		Figure 4  ICBP90 is down-regulated by transcriptional suppression and protein degradation under the control of DNA damage signals. (A) Reduction of mRNA expression of ICBP90 by adriamycin treatment. Expression of ICBP90 and GAPDH mRNAs in HCT116 cells treated with 500 ng/ml adriamycin or 10 µg/ml {alpha}-amanitin for 24 h was determined by semiquantitative RT-PCR analysis, as described in ‘Experimental Procedures’. Cells cultured without drugs were used as controls. (B) Involvement of proteolytic cleavage and/or ubiquitin/proteasome-mediated protein degradation in ICBP90 reduction in adriamycin-treated HCT116 cells. Cells were treated with 500 ng/ml of adriamycin for 24 h in the absence or presence of 10 µM MG132 and immunoblotted with anti-ICBP90 or anti-{alpha}-tubulin antibodies. Cells treated with 10 µg/ml {alpha}-amanitin for 24 h were also analysed.

 
Requirement of p53 for DNA damage-induced ICBP90 reduction

The tumour suppressor protein p53 is a key protein that is activated by the ATM-mediated DNA damage-signalling pathways and that coordinates DNA repair with cell cycle progression and apoptosis. To directly test the role of p53 in regulation of ICBP90 expression after DNA damage, we took advantage of the availability of isogenic HCT116 variant cells, parental (p53+/+) and p53–/– cells (Bunz et al. 1998).

After exposure to adriamycin for 36 h, p53+/+ HCT116 cells arrested both in 2 N G1 and in 4 N G2 (Fig. 5A). In contrast, exposed p53–/– HCT116 cells failed to arrest in 2 N G1 but proceeded to 4 N status (Fig. 5A), indicating that a p53-dependent DNA damage-induced G1 checkpoint is impaired in p53–/– HCT116 cells. Immunoblot analysis showed that adriamycin treatment induced the expression of p53 and p21Cip1/WAF1 and the reduction of ICBP90 in p53+/+ cells (Fig. 5B). In contrast, in p53–/– cells, neither p21Cip1/WAF1 induction nor ICBP90 reduction were observed after DNA damage (Fig. 5B). Immunocytochemical analysis also revealed that ICBP90 was not reduced in adriamycin-treated p53–/– cells which were positive for {gamma}-H2AX staining (Fig. 5C).



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  		Figure 5  p53 is required for ICBP90 down-regulation. (A) HCT116 (p53+/+ and p53–/–) cells and HeLa cells were treated with 500 ng/ml adriamycin for 36 h. Treated and untreated cells were harvested for FACS analysis. (B) Immunoblot analysis of HCT116 (p53+/+ and p53–/–) cells and HeLa cells prepared as described in (A). The protein levels of ICBP90, p53, p21Cip1/WAF1 or ß-actin were determined. (C) Immunocytochemical analysis of HCT116 (p53+/+ and p53–/–) cells and HeLa cells prepared as described in (A). Cells treated with adriamycin were fixed and stained with anti-ICBP90 (FITC, green) and anti-phospho-H2AX (Texas Red, red) antibodies.

 
We performed the same experiments using HeLa cells, in which the p53 is functionally inactivated by human papilloma virus-E6, to confirm the involvement of p53 in ICBP90 expression. HeLa cells also failed to arrest in 2 N G1 and proceeded to 4 N status (Fig. 5A) and maintained a high expression of ICBP90 when DNA damage was induced by adriamycin treatment (Fig. 5B,C). All these findings indicate that the down-regulation of ICBP90 is promoted by p53-dependent, DNA damage-induced signals.

Implication of p21Cip1/WAF1 expression in ICBP90 levels in DNA-damaged cells

P53 activation and stabilization by DNA damage transcriptionally induced expression of p21Cip1/WAF1, which plays a critical role in p53-induced cell cycle arrest. Double staining for ICBP90 and p21Cip1/WAF1 in HCT116 (parental) cells cultured under normal conditions revealed that a small number of cells negative for ICBP90 tended to be positive for p21Cip1/WAF1 (Fig. 6A). Accordingly, we investigated the relationship between p21Cip1/WAF1 induction and ICBP90 reduction after DNA damage. Immunocytochemical analysis revealed that ICBP90 staining was markedly reduced in HCT116 (parental) cells, which were positive for p21Cip1/WAF1 after adriamycin treatment (Fig. 6B). This was consistent with the immunoblot data shown in Fig. 5B. We were able to speculate that p21Cip1/WAF1 is responsible for the reduction of ICBP90 after DNA damage.



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  		Figure 6  P21Cip1/WAF1 plays a critical role in ICBP90 down-regulation. (A) Expression and distribution of ICBP90 and p21Cip1/WAF1 proteins in HCT116 (parental) cells cultured under normal conditions. Cells were subjected to indirect immunofluorescence staining with the anti-ICBP90 (FITC, green) and anti-p21Cip1/WAF1 (Texas Red, red) antibodies. The merged image is shown in right. (B) Immunocytochemical analysis of ICBP90 and p21Cip1/WAF1 in HCT116 (parental) cells treated with adriamycin. HCT116 cells were treated with 500 ng/ml adriamycin for 36 h and were stained with anti-ICBP90 (FITC, green) and anti-p21Cip1/WAF1 (Texas Red, red) antibodies. The merged images are shown in right panels. (C) ICBP90 is down-regulated by adenoviral expression of p21Cip1/WAF1 in HCT116 (p53–/–) cells. P53–/– cells were infected with recombinant adenovirus that expresses luciferase, p21Cip1/WAF1 or p27Kip1 as described in ‘Experimental Procedures’. After 24 h, whole cell extracts were harvested and immunoblotted with indicated antibodies. (D) HCT116 (p21–/–) cells were treated with 500 ng/ml adriamycin for 48 h. Treated and untreated cells were harvested for FACS analysis. Adriamycin-treated p21–/– cells failed to arrest in 2 N G1 but proceeded to 4 N status. (E) Immunoblot analysis of HCT116 (p21–/–) cells prepared as described in (D). The protein levels of ICBP90, p53, p21Cip1/WAF1 or ß-actin were determined. Note that adriamycin treatment induced p53 accumulation but did not promote the down-regulation of ICBP90 in p21–/– cells. (F) Immunocytochemical analysis of HCT116 (p21–/–) cells prepared as described in (D). Cells treated with adriamycin were fixed and stained with anti-ICBP90 (FITC, green) and anti-{gamma}-H2AX (Texas Red, red) antibodies. The merged images are shown in right panels.

 
To test the direct effect of p21Cip1/WAF1 on ICBP90 reduction, we examined ICBP90 levels in HCT116 (p53–/–) cells infected with recombinant adenovirus that expresses p21Cip1/WAF1. Adenoviral expression of p21Cip1/WAF1 led to significant reduction of ICBP90 protein in p53–/– cells without DNA damage (Fig. 6C). In contrast, adenoviral expression of p27Kip1, another cdk inhibitor that induces G1 arrest, did not affect the ICBP90 protein levels (Fig. 6C). The same results were observed when those recombinant proteins were expressed in HCT116 (parental) cells (supplemental figure S2). To further examine the direct role of p21Cip1/WAF1 in ICBP90 expression, we used HCT116 (p21–/–) cells. Immunoblot and immunocytochemical analyses revealed that ICPB90 expression was not changed by adriamycin treatment in p21–/– cells although the p53 levels were increased (Fig. 6D–F). Together, our data suggest that expression of p21Cip1/WAF1 under the control of p53 accumulation after DNA damage promotes the down-regulation of ICBP90.

ICPB90 plays a role in G1/S transition

Np95, a potential murine homolog of ICBP90, has been found to be essential for S phase entry (Bonapace et al. 2002). It is thus possible that down-regulation of ICBP90 by the activation of a p53/p21Cip1/WAF1 pathway plays a role in cell cycle arrest at G1/S transition after DNA damage. To test this hypothesis, we attempted an acute knockdown of ICBP90 by applying of RNA interference (RNAi) in HeLa cells. Cells were transfected with small interfering RNA (siRNA) and treated with adriamycin. Immunoblot analysis performed at 48 h after the transfection of ICBP90 siRNAs (ICBP90–91si, ICBP90-412si and ICBP90–799si) revealed a marked reduction of ICBP90 expression compared to control siRNA (Fig. 7A). Cells transfected with control siRNA failed to arrest at G1 and proceeded to 4 N status after adriamycin treatment because p53 is functionally inactivated in HeLa cells (Fig. 7B); the same result was observed when non-transfected HeLa cells were treated with adriamycin (Fig. 5A). In contrast, a significant fraction of cells transfected with ICBP90 siRNAs (ICBP90–91si and ICBP90–799si) arrested at 2 N G1 phase (Fig. 7B), suggesting that reduction of ICBP90 contributes to G1 arrest induced by DNA-damage-activated checkpoint signals.



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  		Figure 7  ICBP90 contributes to the G1/S transition after DNA double strand breaks. (A) Inhibition of ICBP90 and E2F-1 protein levels by transfection of HeLa cells with siRNAs. After 48 h, whole cell extracts were subjected to immunoblot analysis with antibodies targeted to the indicated proteins. (B) FACS analysis of siRNAs transfected HeLa cells. Cells were transfected with indicated siRNAs for 24 h and then incubated with or without adriamycin (500 ng/ml) for an additional 30 h, when they were harvested for FACS analysis. (Left panel) Depletion of ICBP90 and E2F-1 did not affect DNA histograms of HeLa cells in the absence of adriamycin treatment compared to cells transfected with control siRNA. (Right panel) Adriamycin treatment of control HeLa cells induced 4 N G2 arrest without 2 N G1 arrest. In contrast, 2 N G1 peak appeared in cells depleted ICBP90 or E2F-1.

 
E2F is a key transcriptional factor for G1/S transition. Cdk-mediated hyperphosphorylation of pRb causes release of E2F from pRb-mediated inhibition and advances the cell cycle by transactivating genes required for S phase entry (Duronio et al. 1995; Johnson et al. 1993; Kato et al. 1993; Nevins 1992). It has been reported previously that E2F transactivates the expression of ICBP90 (Mousli et al. 2003), which is consistent with our present data showing that depletion of E2F-1 by siRNA slightly reduced the levels of ICBP90 protein at 48 h after transfection (Fig. 7A, left panels). In contrast, depletion of ICBP90 did not effect of E2F-1 protein levels (Fig. 7A, right panels). A significant number of E2F-1 depleted HeLa cells arrested at G1 when cells were treated with adriamycin, which was very similar to the fate of ICBP90-depleted HeLa cells (Fig. 7B). Therefore, ICBP90 expression plays a crucial role in G1/S transition and, conversely, the down-regulation of ICBP90 by inactivation of E2F may be an important mechanism of G1 arrest when a DNA damage checkpoint is activated.


   Discussion
Top
 Abstract
 Introduction
 Results
 Discussion
Experimental procedures
 Supplementary material
 References
 
Our investigation found that expression of ICBP90 protein is regulated by DNA-damage-activated checkpoint signals. We also identified that p21Cip1/WAF1, which is induced by p53 stabilization after DNA damage, promotes reduction of ICBP90 expression. We here propose that the down-regulation of ICBP90 is an important mechanism for cell cycle arrest at G1/S transition, which is induced by the activation of a p53/p21Cip1/WAF1-dependent DNA-damage checkpoint.

ICBP90 as a target of p53/p21Cip1/WAF1-dependent DNA damage-induced checkpoint

Using the HCT116 cell line (p53+/+) and its p53-deficient variant (p53–/–) we found that ICBP90 expression is controlled by p53-dependent DNA-damage checkpoint signals. Three of our observations support the possibility that p21Cip1/WAF1 induced by p53 activation is directly involved in regulation of ICBP90 levels. First, ICBP90 was not detected in most p53+/+ HCT116 cells expressing p21Cip1/WAF1 by DNA damage signals (Fig. 6B). Second, adenoviral expression of p21Cip1/WAF1 in p53–/– HCT116 cells in the absence of DNA damage down-regulated the expression of ICBP90 (Fig. 6C). Third, the down-regulation of ICBP90 by DNA damage was not observed in p21–/– HCT116 cells (Fig. 6E). P21Cip1/WAF1 is an endogenous inhibitor for the cyclinE/cdk2 complex and, thereby, the expression of p21Cip1/WAF1 maintains pRb in a hypophosphorylated state that sequesters E2F transcriptional factor. ICBP90 expression was recently found to be transcriptionally regulated by E2F (Mousli et al. 2003), and we also confirmed that depletion of E2F-1 by siRNA significantly reduced ICBP90 levels (Fig. 7A). Thus, inactivation of E2F by p21Cip1/WAF1 induction can be a potential mechanism of ICPB90 down-regulation when DNA is damaged. However, inhibition of cyclinE/cdk2 activity by adenoviral expression of p27Kip1, which leads to inactivation of E2F, was not sufficient to reduce the ICBP90 protein (Fig. 6C and supplemental figure S2); while adenoviral expression of p21Cip1/WAF1 effectively down-regulated ICBP90 (Fig. 6C and supplemental figure S2). Treatment with mimosine, which induces high expression of endogenous p27Kip1 and G1 arrest, also did not effect on ICBP90 levels (supplemental figure S3). Furthermore, we found that transcriptional suppression of ICBP90 by {alpha}-amanitin did not induce the rapid down-regulation of ICBP90 protein (Fig. 4) and that proteolytic cleavage and/or ubiquitin/proteasome-dependent protein degradation are potentially involved in ICBP90 down-regulation (Fig. 4B). In fact, we determined that ICBP90 protein is polyubiquitinated in the presence of MG132 (supplemental figure S4). Therefore, the combined data suggest that the DNA damage-induced p21Cip1/WAF1 may activate ICBP90 protein degradation through ubiquitin/proteaosome-mediated pathway or unknown mechanisms in addition to suppression of ICBP90 transcription.

It has been reported previously that Np95 interacts and co-localizes with PCNA in the S phase nucleus (Miura et al. 2001; Uemura et al. 2000), and we confirmed that ICBP90 also partially co-localized with PCNA in human cells (supplemental figure S5). In addition, p21Cip1/WAF1 is known to interact with PCNA during S phase to inhibit DNA replication. Therefore, it is possible that the p21Cip1/WAF1/PCNA complex associates with ICBP90 and may play a role in regulation of ICBP90 protein levels.

Down-regulation of ICBP90 by a DNA damage checkpoint signal contributes to cell cycle arrest at G1/S transition

DNA damage did not induce G1 arrest in HeLa cells, probably due to the fact that p53 is functionally inactivated in those cells (Fig. 5A). However, depletion of ICBP90 expression by siRNA transfection induced G1 arrest in a significant population of HeLa cells when the cells were treated with adriamycin to induce DNA damage. This effect was very similar to that of E2F-1 depletion, which induced the down-regulation of ICBP90 protein at 48 h after the transfection (Fig. 7A). These findings suggest that ICBP90 participates in the G1/S transition and that the down-regulation of ICBP90 by DNA damage signals is a critical factor for G1 arrest.

Recent reports describe the concomitant expression of mouse Np95 and of cyclin E/cdk2 alone as sufficient to induce S phase in terminally differentiated myotube cells (Bonapace et al. 2002). In addition, the expression of Np95 in NIH3T3 cells was tightly regulated during the cell cycle, and its functional impairment resulted in abrogation of DNA synthesis (Sakai et al. 2003). These data support our hypothesis that ICBP90/Np95 family proteins are essential for S phase entry and are important targets of the DNA damage checkpoint.

The important question remaining to be addressed is how ICBP90 regulates S phase entry. ICBP90 has been identified as a transcription factor that plays a role in the TopoII{alpha} gene induction at the G1/S boundary (Hopfner et al. 2000, 2002). TopoII{alpha} is known to be important for induction of genes at the G1/S transition and to promote cell proliferation (reviewed in Muller & Helin 2000). Accordingly, the transcriptional activation of the TopoII{alpha} gene is one of the potential mechanisms by which ICBP90 participates in S phase entry. Future analyses will be required to clarify whether ICBP90 regulates transcription of other genes that are essential for G1/S transition.

The presence in ICBP90 of many structural and functional domains, including a ubiquitin-like domain, a PHD finger, pRb-binding motifs, and a RING-finger domain, suggests that ICBP90 might be involved in protein-protein interactions and enzymatic activities required for S phase entry. Furthermore, as mentioned above, ICBP90 partially co-localizes with PCNA in the S-phase nucleus, suggesting that ICBP90 may not only commit DNA replication by transactivating TopoII{alpha} and other genes but also act as a component in the replication machinery.

Recent observations revealed that Np95-null mouse embryonic stem (ES) cells were more sensitive to DNA damage induced by ionizing radiation, UV light, and N-methyl-N''-nitro-N-nitrosoguanidine (MNNG) than wild-type or Np95+/– cells (Muto et al. 2002). These data, together with our findings, suggest that ICBP90/Np95 functions as a component in the DNA damage response pathways and that it plays a role in the maintenance of genomic stability.


   Experimental procedures
Top
 Abstract
 Introduction
 Results
 Discussion
 Experimental procedures
Supplementary material
 References
 
Cell lines and cell culture

Colon carcinoma cells, parental HCT116 (p53+/+ and p21+/+), p53–/– HCT116 and p21–/– HCT116 (kind gifts from Dr B. Vogelstein), MRC-5 human fibroblasts and HeLa cells were cultured in DMEM/F12 supplemented with 10% foetal calf serum without antibiotics at 37 °C in a humidified atmosphere of 5% CO2.

Cell cycle analysis

For cell cycle analysis and DNA content evaluation, cells were collected by trypsinization, washed with PBS, fixed in cold 70% ethanol, and stored at –20 °C until stained. After fixation, cells were washed twice in PBS and were incubated with 100 µg/ml of RNase A for 30 min at room temperature and stained with 25 µg/ml of propidium iodine (PI) for 30min at room temperature. Flow cytometry was carried out with a FACScalibur flow cytometer and analysed with CellQuest software (Becton Dickinson).

Western blot analysis

Cells were rinsed in PBS, and cells were lysed by loading buffer (2% SDS, 10% glycerol, 50 mM Tris pH 6.8). Samples were subsequently sonicated for 15 s using a microtip. Protein concentration was quantified using Biorad protein assay for all samples lysed in SDS. Cell lysates were denatured at 95 °C for 5 min after the addition of dithiothreitol (DTT) and bromophenol blue. Approximately 25 µg of protein was loaded per lane (except for E2F-1 analysis, where 50 µg of protein were loaded) and separated using a 5–20% gradient polyacrylamide gel (PAGEL, ATTO), and transferred to nitrocellulose membrane (Hybond, Amersham pharmacia). The membranes were probed with primary antibodies. The following antibodies were used: mouse monoclonal antibody for ICBP90 (1RC1C-10, 100 ng/mL) (Hopfner et al. 2000), p53 (DO-1, 1 : 1000 dilution; Santa Cruz), phospho-p53 Ser15 (16G8, 1 : 1000 dilution; Cell Signalling), p27Kip1 (1 : 1000 dilution; Transduction Laboratories), cyclin B (1 : 1000 dilution, Transduction Laboratories), E2F-1 (KH95, 1 : 500 dilution, Santa Cruz), ß-actin (1 : 10 000 dilution; Sigma) and {alpha}-tubulin (1 : 10000 dilution; Sigma). The membranes were then incubated with horseradish peroxidase-conjugated secondary antibodies and visualized by chemiluminescense detection system. For detection of p21Cip1/WAF1 protein, we used horseradish peroxidase-conjugated anti-p21 antibody (C-19 HRP, 1 : 1000 dilution; Santa Cruz).

Immunofluorescence microscopic analysis

Cells were rinsed with TBS buffer (10 mM Tris, pH 7.4, 100 mM NaCl), and prefixed with 3.7% formaldehyde for 2 min at room temperature, washed with TBS, and then fixed with 80% methanol for 5 min. Cells were stained with primary antibody for 1 h at room temperature. The following antibodies were used: mouse monoclonal antibody for ICBP90 (1RC1C-10, 1 g/mL), rabbit polyclonal anti-phospho-Histone H2AX antibody ({gamma}-H2AX, 1 : 100 dilution; TREVIGEN), rabbit polyclonal anti-PCNA antibody (1 : 100 dilution; Santa Cruz) and rabbit polyclonal anti-p21 antibody (C-19, 1 : 100 dilution; Santa Cruz). After a wash with TBS, the cells were stained with fluorescein isothiocyanate (FITC)-conjugated anti-mouse IgG (1 : 200 dilution; Biosource) and PI or Texas Red-conjugated anti-rabbit IgG (1 : 200 dilution; Molecular Probes). The stained cells were mounted with 1,4-diazabicyclo-[2,2,2]-octane/glycerol, and observed with confocal laser scanning microscopy (Fluoview; Olympus).

Semiquantitative RT-PCR analysis

Total RNA was isolated using RNeasy spin column kits (QIAGEN) according to the manufacturer's instructions. The cDNAs were synthesized from 2.5 µg total RNAs with SuperScript II RT (Gibco-BRL). The RT-PCR exponential phase was determined on 22 or 30 cycles to allow semiquantitative comparisons among cDNAs developed from identical reactions. Each PCR regime involved a 95 °C, 5 min initial denaturation step followed by 30 cycles (for ICBP90) or 22 cycles (for GAPDH) at 95 °C for 1 min, 55 °C for 1 min, and 72 °C for 1 min on a GeneAmp PCR system 9700 (Perkin Elmer). The PCR products were separated by electrophoresis on 2% agarose gels.

Adenovirus infection

Three previously described replication-deficient recombinant adenovirus vectors were used in the study (Kanegae et al. 1995; Zhang et al. 1993). Ad5CMV-p21 and Ad5CMV-p27 express the wild-type human p21Cip1/WAF1 and p27Kip1, respectively, under the control of the human cytomegarovirus promoter. Ad5CMV-Luc encoding Luciferase gene was used as a control. Adenoviruses were propageted and titrated in the permissive 293 cell line. The viral infection titres were determined as previously described (Miyake et al. 1998). Cells (3 x 105) were plated 24 h before infection and cultured with adenovirus at 100 MOI for an additional 24 h.

SiRNA transfection

The sequences of the siRNAs were as follows: ICBP90, 5'-AAGATCCAGGAGCTGTTCCAC-3' (corresponding to nucleotides 91–111 relative to the start codon), 5'- AATGAGTACGTCGATGCTCGG-3' (nucleotides 412–432) or 5'-AACGACTGTCGGATCATCTTC-3' (nucleotides 799–819), and E2F1, 5'-AAGTCACGCTATGAGACCTCA-3' (nucleotides 373–393). The 21 nucleotide RNA-DNA chimeric duplexes were obtained from Japan Bioservice (Osaka, Japan). Annealing of the component strands of each siRNA and transfection were performed as described (Elbashir et al. 2001).


   Supplementary material
Top
 Abstract
 Introduction
 Results
 Discussion
 Experimental procedures
 Supplementary material
References
 
The following supplementary figures are available from http://www.blackwellpublishing.com/products/journals/suppmat/GTC/GTC_710/GTC_710sm.htm

Figure S1 The protein levels of ICBP90 in normal human fibroblast, MRC-5 cells treated with 500 ng/mL adriamycin for 48 h. Whole cell extracts were subjected to immunoblot analysis with anti-ICBP90 and anti-{alpha}-tubulin antibodies.

Figure S2 ICBP90 is down-regulated by adenoviral expression of p21Cip1/WAF1 in HCT116 (parental) cells. HCT116 cells were infected with recombinant adenovirus that expresses luciferase, p21Cip1/WAF1 or p27Kip1, as described in ‘Experimental Procedures’. After 24 h, whole cell extracts were harvested for immunoblot analysis.

Figure S3 The protein levels of ICBP90 in HCT116 (parental) cells treated with mimosine at a final concentration of 50 µM to induce G1 arrest. After 48 h, whole cell extracts were subjected to immunoblot analysis with anti-ICBP90 and anti-{alpha}-tubulin antibodies. FACS analysis was performed to confirm that mimosine treatment induced G1 arrest. In control cells, percentages of cells at G1, S and G2/M were 49.7%, 34.9% and 15.4%, respectively. In mimosin treated cells, G1, S and G2/M were 78.2%, 13.1% and 8.7%, respectively.

Figure S4 Polyubiquitination of ICBP90 protein. HCT116 (parental) cells were pre-treated with MG132 for 30 min and then exposed to 500 ng/mL adriamycin. After 24 h, cells were lysed in RIPA buffer supplemented with protease inhibitors. Cell extracts were immunoprecipitated with anti-ICBP90 antibody (ICBP90-IP) and immunoblotted with anti-ubiquitin antibody (P4D1, Santa Cruz). High molecular weight smeary patterns represent ubiquitinated forms of endogenous ICBP90.

Figure S5 Co-localization of ICBP90 and PCNA in HCT116 (parental) cells. Cells cultured under normal conditions were doubly stained with anti-ICBP90 (FITC, green) and anti-PCNA (Texas Red, red) antibodies. The merged images are shown in right panels. Representative S phase cells are shown.


   Acknowledgements
 
We thank Dr B. Vogelstein for providing p53–/– HCT116 cells and p21–/– HCT116 cells; all colleagues in Department of Tumour Genetics and Biology, Graduate School of Medical Sciences, Kumamoto University, Dr Y. Taya (National Cancer Centre) and Mr M. Yoshimoto (Link Genomics, Inc.) for helpful discussion. We are also grateful to members of the Gene Technology Centre and General Research Institute in Kumamoto University for their important contributions to the experiments. This work was supported by a grant for cancer research from the Ministry of Education, Science and Culture of Japan (H. Saya).


   Footnotes
 
Communicated by: Kozo Kaibuchi

* Correspondence: E-mail: hsaya{at}gpo.kumamoto-u.ac.jp


   References
Top
 Abstract
 Introduction
 Results
 Discussion
 Experimental procedures
 Supplementary material
 References
 
Banin, S., Moyal, L., Shieh, S., et al. (1998) Enhanced phosphorylation of p53 by ATM in response to DNA damage. Science 281, 1674–1677.[Abstract/Free Full Text]

Bonapace, I.M., Latella, L., Papait, R., et al. (2002) Np95 is regulated by E1A during mitotic reactivation of terminally differentiated cells and is essential for S phase entry. J. Cell Biol. 157, 909–914.[Abstract/Free Full Text]

Bourdon, J.C., Deguin-Chambon, V., Lelong, J.C., et al. (1997) Further characterisation of the p53 responsive element – identification of new candidate genes for trans-activation by p53. Oncogene 14, 85–94.[CrossRef][Medline]

Bunz, F., Dutriaux, A., Lengauer, C., et al. (1998) Requirement for p53 and p21 to sustain G2 arrest after DNA damage. Science 282, 1497–1501.

Canman, C.E., Lim, D.S., Cimprich, K.A., et al. (1998) Activation of the ATM kinase by ionizing radiation and phosphorylation of p53. Science 281, 1677–1679.[Abstract/Free Full Text]

Chen, I.T., Smith, M.L., O'Connor, P.M. & Fornace, A.J. Jr (1995) Direct interaction of Gadd45 with PCNA and evidence for competitive interaction of Gadd45 and p21Waf1/Cip1 with PCNA. Oncogene 11, 1931–1937.[Medline]

el-Deiry, W.S., Kern, S.E., Pietenpol, J.A., Kinzler, K.W. & Vogelstein, B. (1992) Definition of a consensus binding site for p53. Nature Genet. 1, 45–49.

Duronio, R.J., O'Farrell, P.H., Xie, J.E., Brook, A. & Dyson, N. (1995) The transcription factor E2F is required for S phase during Drosophila embryogenesis. Genes Dev. 9, 1445–1455.[Abstract/Free Full Text]

Elbashir, S.M., Harborth, J., Lendeckel, W., Yalcin, A., Weber, K. & Tuschl, T. (2001) Duplexes of 21-nucleotide RNAs mediate RNA interference in cultured mammalian cells. Nature 411, 494–498.[CrossRef][Medline]

Fujimori, A., Matsuda, Y., Takemoto, Y., et al. (1998) Cloning and mapping of Np95 gene which encodes a novel nuclear protein associated with cell proliferation. Mamm. Genome 9, 1032–1035.[CrossRef][Medline]

Funk, W.D., Pak, D.T., Karas, R.H., Wright, W.E. & Shay, J.W. (1992) A transcriptionally active DNA-binding site for human p53 protein complexes. Mol. Cell. Biol. 12, 2866–2871.[Abstract/Free Full Text]

Hartwell, L.H. & Kastan, M.B. (1994) Cell cycle control and cancer. Science 266, 1821–1828.[Abstract/Free Full Text]

Hartwell, L.H. & Weinert, T.A. (1989) Checkpoints: controls that ensure the order of cell cycle events. Science 246, 629–634.[Abstract/Free Full Text]

Hopfner, R., Mousli, M., Jeltsch, J.M., et al. (2000) ICBP90, a novel human CCAAT binding protein, involved in the regulation of topoisomerase IIalpha expression. Cancer Res. 60, 121–128.[Abstract/Free Full Text]

Hopfner, R., Mousli, M., Oudet, P. & Bronner, C. (2002) Overexpression of ICBP90, a novel CCAAT-binding protein, overcomes cell contact inhibition by forcing topoisomerase II alpha expression. Anticancer Res. 22, 3165–3170.[Medline]

Isaacs, R.J., Davies, S.L., Sandri, M.I., Redwood, C., Wells, N.J. & Hickson, I.D. (1998) Physiological regulation of eukaryotic topoisomerase II. Biochim. Biophys. Acta 1400, 121–137.[Medline]

Jacob, S.T., Sajdel, E.M. & Munro, H.N. (1970) Specific action of alpha-amanitin on mammalian RNA polymerase protein. Nature 225, 60–62.[Medline]

Johnson, D.G., Schwarz, J.K., Cress, W.D. & Nevins, J.R. (1993) Expression of transcription factor E2F1 induces quiescent cells to enter S phase. Nature 365, 349–352.[CrossRef][Medline]

Kanegae, Y., Lee, G., Sato, Y., et al. (1995) Efficient gene activation in mammalian cells by using recombinant adenovirus expressing site-specific Cre recombinase. Nucl. Acids Res. 23, 3816–3821.[Abstract/Free Full Text]

Kato, J., Matsushime, H., Hiebert, S.W., Ewen, M.E. & Sherr, C.J. (1993) Direct binding of cyclin D to the retinoblastoma gene product (pRb) and pRb phosphorylation by the cyclin D-dependent kinase CDK4. Genes Dev. 7, 331–342.[Free Full Text]

Khanna, K.K., Keating, K.E., Kozlov, S., et al. (1998) ATM associates with and phosphorylates p53: mapping the region of interaction. Nature Genet. 20, 398–400.[CrossRef][Medline]

Lakin, N.D. & Jackson, S.P. (1999) Regulation of p53 in response to DNA damage. Oncogene 18, 7644–7655.[CrossRef][Medline]

Lengauer, C., Kinzler, K.W. & Vogelstein, B. (1997) Genetic instability in colorectal cancers. Nature 386, 623–627.[CrossRef][Medline]

Lindell, T.J., Weinberg, F., Morris, P.W., Roeder, R.G. & Rutter, W.J. (1970) Specific inhibition of nuclear RNA polymerase II by alpha-amanitin. Science 170, 447–449.[Abstract/Free Full Text]

Luo, Y., Hurwitz, J. & Massague, J. (1995) Cell-cycle inhibition by independent CDK and PCNA binding domains in p21Cip1. Nature 375, 159–161.[CrossRef][Medline]

Miura, M., Watanabe, H., Sasaki, T., Tatsumi, K. & Muto, M. (2001) Dynamic changes in subnuclear NP95 location during the cell cycle and its spatial relationship with DNA replication foci. Exp. Cell Res. 263, 202–208.[CrossRef][Medline]

Miyake, H., Hanada, N., Nakamura, H., et al. (1998) Overexpression of Bcl-2 in bladder cancer cells inhibits apoptosis induced by cisplatin and adenoviral-mediated p53 gene transfer. Oncogene 16, 933–943.

Mousli, M., Hopfner, R., Abbady, A.Q., et al. (2003) ICBP90 belongs to a new family of proteins with an expression that is deregulated in cancer cells. Br. J. Cancer 89, 120–127.[CrossRef][Medline]

Muller, H. & Helin, K. (2000) The E2F transcription factors: key regulators of cell proliferation. Biochim. Biophys. Acta 1470, M1–M12.[Medline]

Muto, M., Kanari, Y., Kubo, E., et al. (2002) Targeted disruption of Np95 gene renders murine embryonic stem cells hypersensitive to DNA damaging agents and DNA replication blocks. J. Biol. Chem. 277, 34549–34555.[Abstract/Free Full Text]

Nevins, J.R. (1992) E2F: a link between the Rb tumor suppressor protein and viral oncoproteins. Science 258, 424–429.[Abstract/Free Full Text]

Nitiss, J.L., Zhou, J., Rose, A., Hsiung, Y., Gale, K.C. & Osheroff, N. (1998) The bis (naphthalimide) DMP-840 causes cytotoxicity by its action against eukaryotic topoisomerase II. Biochemistry 37, 3078–3085.[CrossRef][Medline]

Paulovich, A.G., Toczyski, D.P. & Hartwell, L.H. (1997) When checkpoints fail. Cell 88, 315–321.[CrossRef][Medline]

Rogakou, E.P., Boon, C., Redon, C. & Bonner, W.M. (1999) Megabase chromatin domains involved in DNA double-strand breaks in vivo. J. Cell Biol. 146, 905–916.[Abstract/Free Full Text]

Rogakou, E.P., Pilch, D.R., Orr, A.H., Ivanova, V.S. & Bonner, W.M. (1998) DNA double-stranded breaks induce histone H2AX phosphorylation on serine 139. J. Biol. Chem. 273, 5858–5868.[Abstract/Free Full Text]

Sakai, A., Kikuchi, Y., Muroi, M., et al. (2003) Overexpression of NP95 mRNA by Tumor Promoters in the Promotion Phase of a Two-Stage BALB/3T3 Cell Transformation Assay. Biol. Pharm. Bull. 26, 347–351.[CrossRef][Medline]

Sherr, C.J. & Roberts, J.M. (1995) Inhibitors of mammalian G1 cyclin-dependent kinases. Genes Dev. 9, 1149–1163.

Uemura, T., Kubo, E., Kanari, Y., Ikemura, T., Tatsumi, K. & Muto, M. (2000) Temporal and spatial localization of novel nuclear protein NP95 in mitotic and meiotic cells. Cell Struct. Funct. 25, 149–159.[CrossRef][Medline]

Waga, S., Hannon, G.J., Beach, D. & Stillman, B. (1994) The p21 inhibitor of cyclin-dependent kinases controls DNA replication by interaction with PCNA. Nature 369, 574–578.

Zhang, W.W., Fang, X., Branch, C.D., Mazur, W., French, B.A. & Roth, J.A. (1993) Generation and identification of recombinant adenovirus by liposome-mediated transfection and PCR analysis. Biotechniques 15, 868–872.[Medline]

Zhou, B.B., Chaturvedi, P., Spring, K., et al. (2000) Caffeine abolishes the mammalian G (2)/M DNA damage checkpoint by inhibiting ataxia-telangiectasia-mutated kinase activity. J. Biol. Chem. 275, 10342–10348.[Abstract/Free Full Text]

Received: 8 October 2003
Accepted: 8 December 2003




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