Chk2-deficient mice exhibit radioresistance and defective p53-mediated transcription
2002; Springer Nature; Volume: 21; Issue: 19 Linguagem: Inglês
10.1093/emboj/cdf506
ISSN1460-2075
AutoresHiroyuki Takai, Kazuhito Naka, Yuki Okada, Miho Watanabe, Naoki Harada, Shinichi Saito, Carl W. Anderson, Ettore Appella, Makoto Nakanishi, Hiroshi Suzuki, Kazuo Nagashima, Hirofumi Sawa, Kyoji Ikeda, Noboru Motoyama,
Tópico(s)Cell death mechanisms and regulation
ResumoArticle1 October 2002free access Chk2-deficient mice exhibit radioresistance and defective p53-mediated transcription Hiroyuki Takai Hiroyuki Takai Department of Geriatric Research, National Institute for Longevity Sciences (NILS), Obu, Aichi, 474-8522 Japan Present address: Laboratory of Cell Biology and Genetics, The Rockefeller University, New York, NY, 10021 USA Search for more papers by this author Kazuhito Naka Kazuhito Naka Department of Geriatric Research, National Institute for Longevity Sciences (NILS), Obu, Aichi, 474-8522 Japan Search for more papers by this author Yuki Okada Yuki Okada Laboratory of Molecular and Cellular Pathology, Hokkaido University Graduate School of Medicine, Gotennba, Shizuoka, 412-8513 Japan CREST, JST, Sapporo, 060-8638 Japan Search for more papers by this author Miho Watanabe Miho Watanabe Research Laboratories, Chugai Pharmaceutical Co. Ltd, Gotennba, Shizuoka, 412-8513 Japan Search for more papers by this author Naoki Harada Naoki Harada Research Laboratories, Chugai Pharmaceutical Co. Ltd, Gotennba, Shizuoka, 412-8513 Japan Search for more papers by this author Shin'ichi Saito Shin'ichi Saito Laboratory of Cell Biology, National Cancer Institute, National Institutes of Health, Bethesda, MD, 20892 USA Present address: Gene Response Section, National Cancer Institute, National Institutes of Health, Bethesda, MD, 20892 USA Search for more papers by this author Carl W. Anderson Carl W. Anderson Biology Department, Brookhaven National Laboratory, Upton, NY, 11973 USA Search for more papers by this author Ettore Appella Ettore Appella Laboratory of Cell Biology, National Cancer Institute, National Institutes of Health, Bethesda, MD, 20892 USA Search for more papers by this author Makoto Nakanishi Makoto Nakanishi Department of Biochemistry, Nagoya City University Medical School, Nagoya, 467-8601 Japan Search for more papers by this author Hiroshi Suzuki Hiroshi Suzuki Research Laboratories, Chugai Pharmaceutical Co. Ltd, Gotennba, Shizuoka, 412-8513 Japan Search for more papers by this author Kazuo Nagashima Kazuo Nagashima Laboratory of Molecular and Cellular Pathology, Hokkaido University Graduate School of Medicine, Gotennba, Shizuoka, 412-8513 Japan CREST, JST, Sapporo, 060-8638 Japan Search for more papers by this author Hirofumi Sawa Hirofumi Sawa Laboratory of Molecular and Cellular Pathology, Hokkaido University Graduate School of Medicine, Gotennba, Shizuoka, 412-8513 Japan CREST, JST, Sapporo, 060-8638 Japan Search for more papers by this author Kyoji Ikeda Kyoji Ikeda Department of Geriatric Research, National Institute for Longevity Sciences (NILS), Obu, Aichi, 474-8522 Japan Search for more papers by this author Noboru Motoyama Corresponding Author Noboru Motoyama Department of Geriatric Research, National Institute for Longevity Sciences (NILS), Obu, Aichi, 474-8522 Japan Search for more papers by this author Hiroyuki Takai Hiroyuki Takai Department of Geriatric Research, National Institute for Longevity Sciences (NILS), Obu, Aichi, 474-8522 Japan Present address: Laboratory of Cell Biology and Genetics, The Rockefeller University, New York, NY, 10021 USA Search for more papers by this author Kazuhito Naka Kazuhito Naka Department of Geriatric Research, National Institute for Longevity Sciences (NILS), Obu, Aichi, 474-8522 Japan Search for more papers by this author Yuki Okada Yuki Okada Laboratory of Molecular and Cellular Pathology, Hokkaido University Graduate School of Medicine, Gotennba, Shizuoka, 412-8513 Japan CREST, JST, Sapporo, 060-8638 Japan Search for more papers by this author Miho Watanabe Miho Watanabe Research Laboratories, Chugai Pharmaceutical Co. Ltd, Gotennba, Shizuoka, 412-8513 Japan Search for more papers by this author Naoki Harada Naoki Harada Research Laboratories, Chugai Pharmaceutical Co. Ltd, Gotennba, Shizuoka, 412-8513 Japan Search for more papers by this author Shin'ichi Saito Shin'ichi Saito Laboratory of Cell Biology, National Cancer Institute, National Institutes of Health, Bethesda, MD, 20892 USA Present address: Gene Response Section, National Cancer Institute, National Institutes of Health, Bethesda, MD, 20892 USA Search for more papers by this author Carl W. Anderson Carl W. Anderson Biology Department, Brookhaven National Laboratory, Upton, NY, 11973 USA Search for more papers by this author Ettore Appella Ettore Appella Laboratory of Cell Biology, National Cancer Institute, National Institutes of Health, Bethesda, MD, 20892 USA Search for more papers by this author Makoto Nakanishi Makoto Nakanishi Department of Biochemistry, Nagoya City University Medical School, Nagoya, 467-8601 Japan Search for more papers by this author Hiroshi Suzuki Hiroshi Suzuki Research Laboratories, Chugai Pharmaceutical Co. Ltd, Gotennba, Shizuoka, 412-8513 Japan Search for more papers by this author Kazuo Nagashima Kazuo Nagashima Laboratory of Molecular and Cellular Pathology, Hokkaido University Graduate School of Medicine, Gotennba, Shizuoka, 412-8513 Japan CREST, JST, Sapporo, 060-8638 Japan Search for more papers by this author Hirofumi Sawa Hirofumi Sawa Laboratory of Molecular and Cellular Pathology, Hokkaido University Graduate School of Medicine, Gotennba, Shizuoka, 412-8513 Japan CREST, JST, Sapporo, 060-8638 Japan Search for more papers by this author Kyoji Ikeda Kyoji Ikeda Department of Geriatric Research, National Institute for Longevity Sciences (NILS), Obu, Aichi, 474-8522 Japan Search for more papers by this author Noboru Motoyama Corresponding Author Noboru Motoyama Department of Geriatric Research, National Institute for Longevity Sciences (NILS), Obu, Aichi, 474-8522 Japan Search for more papers by this author Author Information Hiroyuki Takai1,2, Kazuhito Naka1, Yuki Okada3,4, Miho Watanabe5, Naoki Harada5, Shin'ichi Saito6,7, Carl W. Anderson8, Ettore Appella6, Makoto Nakanishi9, Hiroshi Suzuki5, Kazuo Nagashima3,4, Hirofumi Sawa3,4, Kyoji Ikeda1 and Noboru Motoyama 1 1Department of Geriatric Research, National Institute for Longevity Sciences (NILS), Obu, Aichi, 474-8522 Japan 2Present address: Laboratory of Cell Biology and Genetics, The Rockefeller University, New York, NY, 10021 USA 3Laboratory of Molecular and Cellular Pathology, Hokkaido University Graduate School of Medicine, Gotennba, Shizuoka, 412-8513 Japan 4CREST, JST, Sapporo, 060-8638 Japan 5Research Laboratories, Chugai Pharmaceutical Co. Ltd, Gotennba, Shizuoka, 412-8513 Japan 6Laboratory of Cell Biology, National Cancer Institute, National Institutes of Health, Bethesda, MD, 20892 USA 7Present address: Gene Response Section, National Cancer Institute, National Institutes of Health, Bethesda, MD, 20892 USA 8Biology Department, Brookhaven National Laboratory, Upton, NY, 11973 USA 9Department of Biochemistry, Nagoya City University Medical School, Nagoya, 467-8601 Japan ‡H.Takai and K.Naka contributed equally to this work *Corresponding author. E-mail: [email protected] The EMBO Journal (2002)21:5195-5205https://doi.org/10.1093/emboj/cdf506 PDFDownload PDF of article text and main figures. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions Figures & Info The mammalian Chk2 kinase is thought to mediate ATM-dependent signaling in response to DNA damage. The physiological role of mammalian Chk2 has now been investigated by the generation of Chk2-deficient mice. Although Chk2−/− mice appeared normal, they were resistant to ionizing radiation (IR) as a result of the preservation of splenic lymphocytes. Thymocytes and neurons of the developing brain were also resistant to IR-induced apoptosis. The IR-induced G1/S cell cycle checkpoint, but not the G2/M or S phase checkpoints, was impaired in embryonic fibroblasts derived from Chk2−/− mice. IR-induced stabilization of p53 in Chk2−/− cells was 50–70% of that in wild-type cells. Caffeine further reduced p53 accumulation, suggesting the existence of an ATM/ATR-dependent but Chk2-independent pathway for p53 stabilization. In spite of p53 protein stabilization and phosphorylation of Ser23, p53-dependent transcriptional induction of target genes, such as p21 and Noxa, was not observed in Chk2−/− cells. Our results show that Chk2 plays a critical role in p53 function in response to IR by regulating its transcriptional activity as well as its stability. Introduction Disruption of the mechanisms in multicellular organisms that regulate checkpoint and apoptotic responses leads to genomic instability and the development of cancer. Several protein kinases play key roles in the activation of these pathways in response to DNA damage including ataxia-telangiectasia-mutated (ATM) defects that result in ataxia telangiectasia, ATR (ATM and rad3-related) and their kinase substrates Chk2 and Chk1 (Giaccia and Kastan, 1998; Appella and Anderson, 2001). Mammalian Chk1 is essential for early embryonic development and the G2 checkpoint response to DNA damage and replication block (Liu et al., 2000; Takai et al., 2000). Chk2, the mammalian homolog of the Saccharomyces cerevisiae Rad53 and Schizosaccharomyces pombe Cds1 kinases, also is implicated in the DNA damage signaling pathway (Bartek et al., 2001). Chk2 is activated by phosphorylation in an ATM-dependent manner in response to ionizing radiation (IR), and in an ATM-independent manner in response to UV radiation or stalled DNA replication. Activated Chk2 phosphorylates Cdc25A on serine (Ser)123, Cdc25C on Ser215, BRCA1 on Ser988, and p53 on several sites including Ser20 (Bartek et al., 2001). Recently, heterozygous germline mutations in the human Chk2 gene were found in a subset of patients with Li–Fraumeni syndrome (Bell et al., 1999). Most cases of this highly penetrant familial cancer syndrome result from inheritance of a mutant p53 allele and the subsequent somatic loss of the remaining wild-type allele. The p53 tumor suppressor protein plays a central role in a cells' decision to induce either cell cycle arrest or apoptosis after diverse stresses, including DNA damage, hypoxia and the activation of oncogenes (Giaccia and Kastan, 1998; Prives and Hall, 1999; Vousden, 2000). Regulation of the abundance and transcriptional activity of p53 is achieved primarily by post-translational modifications, such as phosphorylation and acetylation (Appella and Anderson, 2001). In normal cells p53 protein levels are low due to Mdm2-mediated ubiquitylation and degradation through the proteasome pathway. Mdm2 may also regulate p53 activity by facilitating nuclear export and degradation following ubiquitylation (Liang and Clarke, 2001). In response to stress such as IR, human p53 is phosphorylated at several sites in its transactivation domain including Ser15 and Ser20. ATM phosphorylates p53 on Ser15 (Banin et al., 1998; Canman et al., 1998), and this phosphorylation was suggested to inhibit the interaction of p53 with Mdm2, resulting in p53 stabilization (Shieh et al., 1997; Chehab et al., 1999). ATM-dependent phosphorylation of Mdm2 reduces its capability to promote nucleo-cytoplasmic shuttling and the subsequent degradation of p53 (Maya et al., 2001). Transcriptional activation of p53 also is regulated by acetylation at several C-terminal lysine (Lys) residues by CBP/p300 and PCAF (Appella and Anderson, 2001). Acetylation is believed to activate the sequence-specific DNA binding ability of p53, which is required for most, if not all, p53-mediated responses (Ito et al., 2001). Data from mutants indicate that acetylation is induced by phosphorylation of sites in the N-terminal transactivation domain of p53, including Ser15, which is thought to recruit CBP/p300 to p53. In addition, ATM was shown to activate Chk2, which phosphorylates several N-terminal p53 sites including Ser20, and these phosphorylations also may contribute to both stabilization and transcriptional activation of the p53 protein (Chehab et al., 2000; Hirao et al., 2000; Shieh et al., 2000). The extent to which Chk2 participates in p53 stabilization and activation in response to DNA damage, such as that induced by IR, remains unclear, however. To characterize the function of Chk2, we generated Chk2-deficient (Chk2−/−) mice. We now show that, in contrast to Chk1-deficient (Chk1−/−) mice (Liu et al., 2000; Takai et al., 2000), Chk2−/− mice are viable and fertile. Moreover, Chk2−/− mice were more resistant than Chk2+/+ mice to sublethal doses of IR. Partial stabilization of the p53 protein was observed in Chk2−/− cells in response to IR; however, even though phosphorylation and acetylation of p53 after IR were apparently normal, the transcriptional activity of p53 was abolished. Thus, Chk2 plays a pivotal role in the biological activity of p53 by regulating its transcriptional activation as well as its stabilization after IR-induced damage. Results Inactivation of the Chk2 gene in mice We generated Chk2−/− mice by gene targeting in embryonic stem (ES) cells (see Supplementary data available at The EMBO Journal Online). Chk2−/− animals were born in the expected ratio and appeared normal. Three of 34 Chk2−/− mice, one of 23 Chk2+/− mice and none of 22 Chk2+/+ mice were dead by 1 year. One of the Chk2−/− mice that died developed a mammary gland epithelial tumor (data not shown). Increased survival of Chk2−/− mice upon IR Chk2 is phosphorylated and activated by ATM in response to IR (Bartek et al., 2001). Given that Atm−/− mice manifest extreme sensitivity to IR (Barlow et al., 1996), we examined whether Chk2 deficiency also results in acute radiation toxicity. Eight to 16-week-old Chk2+/+, Chk2+/− and Chk2−/− mice were exposed to a sublethal dose (8 Gy) of IR and then monitored for survival and illness. Both Chk2+/+ and Chk2+/− mice began to die from 6 days after IR, and more than two-thirds of these animals died within 2 weeks after IR. In sharp contrast, Chk2−/− mice were resistant to IR, and approximately two-thirds survived beyond 30 days after IR exposure (Figure 1A). The 50% survival times for Chk2+/+, Chk2+/− and Chk2−/− mice were 12 ± 0.5 days (means ± standard deviation), 13 ± 1.5 days and >30 days, respectively. Kaplan–Meier survival analysis revealed that the prolongation of survival in Chk2−/− mice was statistically significant (P < 0.0001) compared with Chk2+/+ and Chk2+/− mice. Although Atm−/− mice die within 3–5 days post-irradiation as a result of acute radiation toxicity to the gastrointestinal tract, with epithelial crypt degeneration and abscess formation (Barlow et al., 1996), Chk2−/− mice did not exhibit any pathological abnormalities in the esophagus, stomach and small or large intestines when compared with Chk2+/+ mice after IR (Figure 1B and C). The time of death in Chk2+/+ mice, between 1 and 2 weeks after irradiation, is consistent with the kinetics of lymphocyte depletion and resulting infection, suggesting that Chk2 deficiency might render lymphocytes radioresistant. To evaluate this hypothesis, we performed histological analysis of the spleen after IR. Marked atrophy of, and a reduced number of, lymphocytes in the white pulps were apparent in the spleens of Chk2+/+ and Chk2+/− mice 8 days after IR (Figure 1D; data not shown). In contrast, no such changes were detected in Chk2−/− mice (Figure 1E). These results indicate that lymphocytes in the spleens of Chk2−/− mice are resistant to IR, rendering the mice themselves radioresistant also. Figure 1.Reduced radiosensitivity of Chk2−/− mice and Chk2−/− splenocytes in vivo. (A) Kaplan–Meier survival curve of age-matched 8–16-week-old Chk2+/+(n = 23), Chk2+/− (n = 37) and Chk2−/− (n = 36) mice after exposure to 8 Gy of X-rays. Data are combined from two separate experiments. (B and C) Normal appearance of intestine in Chk2+/+ (B) and Chk2−/− (C) mice 1 day after IR. (D and E) Atrophy of white pulp of the spleen and reduction of splenocyte number in Chk2+/+ mice (D), but not in Chk2−/− mice (E), 8 days after exposure to IR. Download figure Download PowerPoint Decreased cell death in response to IR in Chk2−/− mice The resistance of Chk2−/− splenocytes to IR suggests that Chk2 might be involved in the apoptotic pathway in vivo. Both immature thymocytes and neurons in the developing CNS are susceptible to IR-induced apoptosis (Clarke et al., 1993; Lowe et al., 1993; Herzog et al., 1998). To examine the role of Chk2 in the apoptotic response, we examined the thymus and developing brain of Chk2−/− mice by TUNEL analysis. Although whole body irradiation resulted in widespread apoptosis in the cortical region of the thymus in Chk2+/+ mice (Figure 2A and C), apoptotic cells were rarely detected in the thymus of Chk2−/− mice (Figure 2B and D). Flow cytometric analysis of thymocytes isolated from mice subjected (or not) to IR revealed that CD4 and CD8 double-positive cells from Chk2+/+ mice were highly sensitive to IR, whereas such double-positive thymocytes from Chk2−/− mice were resistant to IR-induced apoptosis (Figure 2M). Consistent with these in vivo observations, Chk2−/− thymocytes irradiated in vitro also were resistant to IR-induced apoptosis (Figure 2N). In the developing CNS of Chk2+/+ mice, neurons in the external germinal layer of the cerebellum and in the dentate gyrus of the hippocampus exhibited widespread apoptotic death in response to IR (Figure 2E, G, I and K). In contrast, neurons in the same regions of the CNS of Chk2−/− mice were resistant to IR-induced apoptosis (Figure 2F, H, J and L). These results indicate that Chk2 mediates IR-induced apoptosis in thymocytes and in neurons of the developing CNS. Figure 2.Defective IR-induced apoptosis in the thymus and developing brain of Chk2−/− mice. (A–D) TUNEL staining of thymi derived from 2- to 3-month-old Chk2+/+ (A and C) and Chk2−/− (B and D) mice 9 h after exposure to 8 Gy of IR. Magnification: 4× (A and B) or 40× (C and D). (E–L) TUNEL staining of the cerebellum from Chk2+/+ (E and G), Chk2−/− (F and H) mice and of the hippocampus from Chk2+/+ (I and K) and Chk2−/− (J and L) mice at 4 days of age and 9 h after exposure to 8 Gy of IR. Magnification: 10× (E, F, I and J) or 40× (G, H, K and L). (M) Flow cytometric analysis of thymocytes isolated from control Chk2+/+ and Chk2−/− mice or from irradiated animals 24 h after exposure to 4 Gy of IR. The cells were stained for CD4 and CD8. (N) Sensitivity of isolated thymocytes to IR. Thymocytes derived from Chk2+/+ and Chk2−/− mice were exposed to 4 Gy of IR and cultured for 24 h before determining viability by staining with propidium iodide and flow cytometry. Data are expressed as a percentage of the viability of the corresponding non-irradiated cells and are means ± standard deviation from three independent experiments. Download figure Download PowerPoint Cell cycle checkpoint in Chk2-deficent cells To examine the role of Chk2 in cell cycle checkpoint responses to IR, cells were irradiated and then mitotic indexes were determined. Within 0.5 h of exposure to IR, the mitotic index had significantly decreased (Figure 3A and B), and the cells accumulated in G2/M, reaching a peak at 6 h, after which the index decreased for both MEFs and ES cells from Chk2+/+ and Chk2−/− mice (Figure 3C and D). To examine maintenance of the G2 checkpoint, irradiated or non-irradiated cells were treated with nocodazole, which traps cells in mitosis, and the mitotic index was then determined at various times. The mitotic index of both non-irradiated Chk2+/+ and Chk2−/− MEFs and ES cells increased with time. In response to IR, both Chk2+/+ and Chk2−/− MEFs and ES cells arrested in G2 for 6 h, after which the proportion of mitotic cells of both genotypes gradually increased with similar kinetics (Figure 3E and F). These data indicate that both the initiation and maintenance of the G2 checkpoint are normal in Chk2−/− MEFs and ES cells. Figure 3.Normal G2/M checkpoint activation in Chk2−/− mice. (A and B) Activation of the G2/M checkpoint. Mitotic index of irradiated or unirradiated Chk2+/+ (squares) and Chk2−/− (circles) MEFs (A) and ES cells (B) was determined at the indicated times after 10 Gy IR. Data are expressed as the percentage of mitotic cells of total cells; means ± standard deviation are from triplicate experiments. (C and D) The fraction of G2/M Chk2+/+ (squares) or Chk2−/− (circles) MEFs (C) or ES cells (D) was determined at the indicated times after 10 Gy IR. The means ± standard deviation from three independent experiments are given. (E and F) Maintenance of the G2/M checkpoint. Irradiated (closed symbols) and unirradiated (open symbols) Chk2+/+ (squares) and Chk2−/− (circles) MEFs (E) and ES cells (F) were treated with 0.2 μg/ml nocodazole, and the percentage of mitotic cells was determined at the indicated times after 10 Gy IR. Means ± standard deviation from replicate (n = 4) (E) or triplicate (F) experiments are given. Download figure Download PowerPoint To examine the G1/S checkpoint in Chk2−/− MEFs, asynchronous MEFs were irradiated and the numbers of cells in G1, S and G2/M were determined by FACS analysis as shown in Figure 4A. For early time points after IR (up to 2 h), the number of S phase cells was the same for both Chk2+/+ and Chk2−/− MEFs (Figure 4B). However, from 4 h after IR, the decrease in the number of Chk2−/− S phase MEFs was smaller than that for Chk2+/+ MEFs (Figure 4B). To examine maintenance of the G1 checkpoint at later times, asynchronous MEFs were treated with nocodazole with or without IR, and then the DNA content was determined as shown in Figure 4C. At 18 and 24 h after IR, the fraction of Chk2+/+ MEFs in the G1 phase was increased, whereas that of Chk2−/− MEFs was not (Figure 4C and D). Finally, serum-starved MEF cells were irradiated (or not) and then stimulated to enter the cycle by the addition of serum. BrdU was also added to the incubation medium to allow the detection of cells entering S phase. Cells were harvested 24 h after stimulation and the number of cells that entered S phase was determined by flow cytometry. Whereas irradiation reduced the proportion of Chk2+/+ cells in S phase by 52%, it reduced the proportion of Chk2−/−cells that had entered S phase by only 25% (Figure 4E). As initiation and maintenance of the G1 checkpoint are regulated, in part, through inhibition of cyclin E-associated Cdk2 activity (Bartek et al., 2001), we examined whether Chk2 deficiency affected this kinase activity after IR. Consistent with our FACS data, cyclin E-associated Cdk2 activities were decreased in both Chk2+/+ and Chk2−/− MEFs in response to IR (Figure 4G and H); however, in Chk2−/− MEFs, its activities again increased by 2.5 h after exposure to IR (Figure 4H). Together, these results show that initiation of the G1 arrest in response to IR, which is independent of p53, is intact, but maintenance of the G1 arrest beyond 2 h, which is dependent on p53, is defective in Chk2−/− MEFs. Figure 4.Defective G1, but not S phase, checkpoint activation in Chk2−/− mice. (A) Representive data of dot-plots of BrdU fluorescence versus DNA content for Chk2+/+ or Chk2−/− MEFs at the times indicated after 10 Gy IR. Irradiated and unirradiated MEFs were treated with 10 μM BrdU for 30 min before cells were harvested and analyzed by flow cytometry. (B) The percentage of total cells that were BrdU positive from Chk2+/+ (squares) or Chk2−/− (circles) MEFs as described in (A); the means ± standard deviation are from triplicate experiments. (C) Representative histograms show DNA content of Chk2+/+ or Chk2−/− MEFs at the times indicated after 10 Gy IR. Cells were treated with 1 mg/ml nocodazole, and at the indicated time points after IR, they were harvested and analyzed by flow cytometry. (D) A summary of triplicate experiments as described in (C). The percentage increase in G1 is the difference in the percent G1 content between irradiated and unirradiated Chk2+/+ (squares) or Chk2−/− (circles) control cells, respectively. (E) Defective ability of Chk2−/− MEFs to block S phase entry following 20 Gy IR. Serum-starved MEFs were released from G1 arrest into complete medium containing BrdU (65 μM) and immediately subjected (or not) to irradiation. Cells were harvested 24 h after release and the number of BrdU positive cells was determined as a percentage of total cells by flow cytometry. Data are means ± standard deviation of values from replicate experiments (n = 4). (F) Normal S phase checkpoint activation in Chk2−/− MEFs. Replicative DNA synthesis was assessed 1 h after the indicated doses of IR in Chk2+/+ (squares) or Chk2−/− (circles) MEFs. (G and H) The activity of cyclin E-associated Cdk2 at 2 h after IR at the indicated dose (Gy) or after 15 J/m2 UV and (G) at the times indicated after IR (20 Gy) (H). Relative kinase activities are indicated at the bottom. Download figure Download PowerPoint ATM-deficient cells undergo radioresistant DNA synthesis (RDS), indicating a defect in IR-induced S phase arrest (Rotman and Shiloh, 1999; Kastan and Lim, 2000). Chk2 phosphorylates Cdc25A in response to IR, inducing its degradation and inhibition of cyclin E-associated Cdk2 kinase activity, which results in S phase arrest (Bartek et al., 2001). To examine the S phase checkpoint in Chk2−/− MEFs, thymidine incorporation was determined in asynchronous MEFs irradiated with X-rays from 4–20 Gy. While inhibition of DNA synthesis did take place in the absence of Chk2, a subtle difference was observed between Chk2+/+ and Chk2−/− MEFs (Figure 4F). Accordingly, inhibition of cyclin E-associated Cdk2 activities was observed in both Chk2+/+ and Chk2−/− MEFs in response to IR (Figure 4G and H). These results indicate that, while Chk2 may play some role in regulating S phase progression, it is dispensable for the overall activation of the S phase checkpoint after IR. Stabilization of p53 protein in Chk2−/− cells after IR The abnormalities in apoptosis and cell cycle checkpoint control in Chk2−/− cells are suggestive of a functional interaction between Chk2 and p53. To better define the role played by Chk2 in p53 stabilization, we analyzed p53 protein levels in Chk2−/− thymocytes, MEFs and ES cells following DNA damage. Consistent with previous observations (Clarke et al., 1993; Lowe et al., 1993), IR induced a marked increase in the amount of p53 in Chk2+/+ thymocytes that was apparent within 1 h (Figure 5A). Accumulation of p53 also was detected in Chk2−/− thymocytes, although the maximum level was only 50–70% of that apparent in Chk2+/+ thymocytes (Figure 5A and B). Similar results were obtained with MEFs (Figure 5C) and ES cells (Figure 5D). Stable expression of human Chk2 in Chk2−/− ES cells restored IR-induced stabilization of p53 to the level apparent in Chk2+/+ ES cells (Figure 5D). These results indicate that Chk2 contributes to the stabilization of p53 induced by IR in vivo. The partial nature of the defect in IR-induced p53 stabilization observed in Chk2-deficient cells suggested the existence of an alternative pathway for p53 stabilization. We therefore examined whether ATM or ATR contribute to p53 stabilization in Chk2−/− cells by investigating the effect of caffeine, a known inhibitor of ATM and ATR (Sarkaria et al., 1999). The extent of IR-induced stabilization of p53 in both Chk2−/− and Chk2+/+ cells was markedly reduced by caffeine (Figure 5E), suggesting that p53 stabilization in Chk2−/− cells is mediated by the ATM or ATR pathways. One of the major downstream effectors of ATR is Chk1 (Liu et al., 2000); thus, we examined the activation of Chk1 after IR. Chk1 exhibited a lower electrophoretic mobility and increased phosphorylation on Ser345 in response to either IR or UV in both Chk2+/+ and Chk2−/− MEFs, but the changes were weaker in response to IR (Figure 5F). These results indicate that Chk1 is activated in response to both types of DNA damage, and that Chk1 may be involved in the regulation of p53 stabilization and function in Chk2−/− cells. Figure 5.IR-induced stabilization of p53 in Chk2−/− thymocytes, MEFs and ES cells. (A) Immunoblot analysis of the time course of p53 abundance after exposure of thymocytes from Chk2+/+, Chk2−/− and p53−/− mice to IR (5 Gy). (B) Quantitation of p53 abundance 4 h after exposure of Chk2+/+ or Chk2−/− thymocytes to IR as in (A). Data are expressed as fold induction relative to the amount of p53 in non-irradiated cells and are means ± standard deviation of values from five independent experiments. (C) Protein levels of p53 in MEFs from Chk2+/+ or Chk2−/− mice harvested at the indicated times after 10 Gy IR. (D) Effect of expression of recombinant human Chk2 on the IR-induced stabilization of p53 in Chk2−/− ES cells. (E) Stabilization of p53 protein after IR is dependent on a caffeine-sensitive pathway in Chk2−/− cells. Chk2+/+ and Chk2−/− ES cells were pre-treated with or without 5 mM caffeine for 1 h and cell lysates were made 2 h after IR. (F) Activation of Chk1 in response to IR. Immunoblot analysis of the time course of Chk1 and Ser345-phosphorylated Chk1 in Chk2+/+ or Chk2−/− MEFs after IR (10 Gy) or UV irradiation (50 J/m2). Download figure Download PowerPoint Defective transcriptional activation of p53 protein in Chk2−/− cells The p53 protein regulates the expression of many genes whose products play important roles in cell cycle arrest and apoptosis. In this work we examined the expression of several known p53 target genes, including Mdm2, Cdkn1a (p21), Pmaip1 (Noxa), Ccng (cyclin G1) and Bax, in thymocytes and MEFs using real-time, quantitative RT–PCR. Messenger RNAs for Mdm2, Cdkn1a, Pmaip1 and Bax were significantly induced with a peak at 4–6 h after IR in Chk2+/+ thymocytes, however, none of these mRNAs accumulated after IR-treatment in Chk2−/− thymocytes (Figure 6A). Similarly, increased mRNA levels for Mdm2, Cdkn1a, Pmaip1 and Ccng were not observed in Chk2−
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