Rhythmic Control of the ARF-MDM2 Pathway by ATF4 Underlies Circadian Accumulation of P53 in Malignant Cells

Published OnlineFirst April 11, 2013; DOI: 10.1158/0008-5472.CAN-12-2492

Cancer Tumor and Stem Cell Biology Research

Rhythmic Control of the ARF-MDM2 Pathway by ATF4 Underlies Circadian Accumulation of p53 in Malignant Cells

Michiko Horiguchi1,2, Satoru Koyanagi1, Ahmed M. Hamdan1, Keisuke Kakimoto1, Naoya Matsunaga1, Chikamasa Yamashita2, and Shigehiro Ohdo1

Abstract The sensitivity of cancer cells to chemotherapeutic agents varies according to circadian time. Most chemotherapeutic agents ultimately cause cell death through cell-intrinsic pathways as an indirect conse- quence of DNA damage. The p53 tumor suppressor gene (TRP53) configures the cell deaths induced by chemotherapeutic agents. In this study, we show that the transcription factor ATF4, a component of the mammalian circadian clock, functions in circadian accumulation of p53 protein in tumor cells. In murine fibroblast tumor cells, ATF4 induced the circadian expression of p19ARF (Cdkn2a). Oscillation of p19ARF interacted in a time-dependent manner with MDM2, a specific ubiquitin ligase of p53, resulting in a rhythmic prevention of its degradation by MDM2. Consequently, the half-life of p53 protein varied in a circadian time- dependent manner without variation in mRNA levels. The p53 protein accumulated during those times when the p19ARF–MDM2 interaction was facilitated. Notably, the ability of the p53 degradation inhibitor nutlin-3 to kill murine fibroblast tumor cells was enhanced when the drug was administered at those times of day during which p53 had accumulated. Taken together, these results suggested that ATF4-mediated regulation of the p19ARF–MDM2 pathway underlies the circadian accumulation of p53 protein in malignant cells. Furthermore, they suggest an explanation for how the sensitivity of cancer cells to chemotherapeutic agents is enhanced at those times of day when p53 protein has accumulated, as a result of circadian processes controlled by ATF4. Cancer Res; 73(8); 2639–49. 2013 AACR.

Introduction Circadian oscillations in biological function are associ- fi Daily rhythmic variations in biological functions are thought ated with time-dependent changes in the ef cacy and/or to affect the efficacy and/or toxicity of drugs: the potency of a toxicity of many drugs. Recent molecular studies of the large number of drugs varies depending on the time of day circadian biological clock system have revealed that oscilla- fi when the drugs are administered (1). It has been suggested that tions in the transcription of speci cclockgenesplaya – administering drugs at appropriate times of day can improve central role in the generation of circadian rhythms (3 5). In the outcome of pharmacotherapy by maximizing potency and mammals, 24-hour rhythms in different tissues are coordi- minimizing the toxicity of the drug, whereas administering nated by a master clock located in the suprachiasmatic them at inappropriate times of day can induce severe side nuclei of the anterior hypothalamus. The master circadian effects (2). A chronopharmacological strategy can also improve clock follows a daily light/dark cycle and, in turn, synchro- tumor response, overall survival, and dose-limiting toxicity in nizes subsidiary oscillators in other brain regions and many cancer patients who are subjects in ongoing prospective peripheral tissues through neural and/or hormonal signals – randomized clinical trials. (6 8). These subsidiary oscillators coordinate a variety of biological processes, producing circadian rhythms in physiology and behavior. Authors' Affiliations: 1Department of Pharmaceutics, Graduate p53 protects cells from uncontrolled growth through acti- School of Pharmaceutical Sciences, Kyushu University, Fukuoka; and vating genes that induce cell-cycle arrest, DNA repair, and 2Faculty of Pharmaceutical Sciences, Tokyo University of Science, Chiba, Japan apoptotic cell death (9, 10). Because of its role in conserving stability by preventing genome mutation, p53 is known as "the Note: Supplementary data for this article are available at Cancer Research Online (http://cancerres.aacrjournals.org/). guardian of the genome." Under nonstress conditions, the protein levels of p53 are tightly regulated by the E3 ubiquitin Current address for A. M. Hamdan: Max-Plank Institute for Biophysical – Chemistry, Am Faßberg 11 D-37077, Gottingen,€ Germany. ligase MDM2 at posttranscriptional level (11 13). When cells are exposed to genotoxic stress such as chemotherapeutic Corresponding Author: Shigehiro Ohdo, Department of Pharmaceutics, Graduate School of Pharmaceutical Sciences, Kyushu University, Fukuoka treatments, p53 proteins are rapidly accumulated via a de- 812-8582, Japan. Phone: 81-92-642-6610; Fax: 81-92-642-6614; E-mail: crease in their degradation (14). Although previous reports [email protected] show that the protein levels of p53 exhibit a significant cir- doi: 10.1158/0008-5472.CAN-12-2492 cadian oscillation in the human oral epithelium and mouse 2013 American Association for Cancer Research. tissues (15, 16), it remains to be clarified whether the oscillation

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of p53 protein levels affects the susceptibility of tumor cells to Determination of p53 protein stability chemotherapeutic agents. To determine the stability of p53 protein in cultured UV. Activating transcription factor (ATF)/cAMP response BAL-5.4G cells, 50 mg/mL of cycloheximide (CHX) was added to element (CRE)-binding (CREB) proteins induce the CRE- media and cell lysates were prepared at selected time intervals. mediated gene transcription depending on the cAMP stim- Cells were lysed in 25 mmol/L Tris-HCl, pH 8.0, 137 mmol/L ulation. Extensive activation of cAMP-dependent signaling is NaCl, 2.7 mmol/L KCl, 1% Triton X-100 supplemented with often observed in malignant tumor cells, which probably protease inhibitor mixtures and proteasome inhibitor (100 contributes to abnormal cell proliferation. cAMP-dependent nmol/L; MG132). To investigate the time-dependent difference signaling oscillates in a circadian time-dependent manner, in the stability of p53 protein in vivo, tumor masses formed by which in turn also sustains core oscillation machinery of the oncogenic wild-type or Atf4 / cells were removed from circadian clock (17). Among the ATF/CREB family proteins, tumor-bearing mice at ZT2 and ZT14, and single-cell suspen- ATF4 constitutes a molecular link connecting cAMP-depen- sion was prepared by standard methods (19). Cells were dent signaling to the circadian clock (18). In this study, we incubated in the presence of 50 mg/mL of CHX. After incuba- showed that ATF4 caused the circadian accumulation of tion, nuclear fractions were extracted using NE-PER Nuclear p53 protein in tumor cells. ATF4 repressed the expression and Cytoplasmic Extraction Reagents (Pierce Biotechnology), of p19ARF in a circadian time-dependent manner; the and they were immunoprecipitated with anti-p53 antibodies oscillation of p19ARF resulted in periodic prevention of (PAb246; Calbiochem). The precipitants were analyzed by MDM2-mediated degradation of p53 protein. We thus inves- Western blotting using a polyclonal anti-p53 antibody (sc tigated how the circadian accumulation of p53 protein 6243; Santa Cruz Biotechnology Co., Ltd.). affects the susceptibility of tumor cells to chemotherapeutic agents. Western blotting Cell lysates were prepared from tumor masses or cultured Materials and Methods cells were extracted as described earlier. The protein concen- Animals and cells trations were determined using a BCA Protein Assay Kit (Pierce Male Balb/c Nu/Nu mice were purchased from Charles Biotechnology). The lysate samples were separated on 5%, 10%, þ River. Heterozygous Atf4-null (Atf4 / ) mice were provided or 15% SDS-polyacrylamide gels and transferred to polyviny- by Dr. S. Akira (Osaka University, Osaka, Japan). The animals lidene difluoride membranes. The membranes were reacted were housed in a temperature-controlled (24 1C) room with antibodies against p53 (sc-6243), ATF4 (sc-200), p19ARF under a 12-hour light : 12-hour dark cycle. Under the light/dark (sc-22784), MDM2 (sc-812), or ACTIN (sc-1616). These anti- cycle, zeitgeber time (ZT) 0 was designated as lights on and bodies were purchased from Santa Cruz Biotechnology. Anti- ZT12 as lights off. The animals were cared in accordance with glutathion S-transferase (GST) antibodies (Wako Co., Ltd.) the guidelines established by the Animal Care and Use Com- were used to detect recombinant GST-fused p53 (rGST-p53) mittee of Kyushu University (Fukuoka, Japan). protein. The immunocomplexes were further reacted with Mouse fibroblast-like tumor cell lines UV.BAL-5.4G horseradish peroxidase–conjugated secondary antibodies. The (RCB2025) were provided by RIKEN Bio Resource Center membranes were photographed and the density of each band (Tsukuba, Japan). The cells are established by UV irradiation, was analyzed by using LAS4000 mini (Fiji film). and were maintained in Dulbecco's Modified Eagle Medium (DMEM; Sigma-Aldrich) supplemented 10% FBS at 37Cina Quantitative RT-PCR analysis humidified 5% CO2 atmosphere. The tumor cell lines expressed Total RNA was extracted using RNAiso reagent (Takara Co., wild-type p53 protein (Supplementary Fig. S1). To assess the Ltd.). The complementary DNA (cDNA) was synthesized by circadian oscillation of p53 expression in cultured cells, semi- reverse transcribing 0.4 mg of RNA using a ReverTra Ace qPCR confluent cultured UV.BAL-5.4G cells were stimulated with 10 RT kit (Toyobo). The cDNA equivalent of 12 ng of RNA was nmol/L dexamethasone (DEX) for 2 hours; the medium was amplified by PCR in a real-time PCR system (Applied Biosys- then replaced with DMEM supplemented with 2% FBS. Cells tems, Life Technologies). Sequences for PCR primers are were harvested for extraction of protein or RNA at the indi- described in Supplementary Table S1. cated times after DEX stimulation. Mouse embryonic fibroblasts (MEF) were prepared by standard techniques from Vector construction embryos of wild-type or Atf4-null (Atf4 / ) mice, and cells Specific silencing of the endogenous Atf4 in tumor cells was were maintained in DMEM supplemented with 10% FBS, 20 achieved by using a shRNA-expressing retrovirus vector. mmol/L b-mercaptoethanol, and 1 nonessential amino acid Nucleotides 1114–1132 of the mouse Atf4 (NM_009716) coding mix. For oncogenic transformation, MEFs were infected with sequence were chosen as the target for shRNA. The Atf4- retroviral vectors expressing H-rasV12 and SV40LT (19). shRNA–encoding oligonucleotides were created as indicated Balb/c Nu/Nu mice were inoculated with UV.BAL-5.4G below, each containing the 19-nucleotide target sequence of (2 107 cells/mouse) or oncogenic-transformed MEFs Atf4, followed by a short spacer and an antisense sequence of (2.5 106 cells/mouse). Tumor volume was measured as the target: 50-GAGCATTCCTTTAGTTTAGAGAGCTCACCC- described previously (19), and tumor-bearing mice were used TAAACTAAAGGAA-TGCTC-30. The Atf4-shRNA–encoding for each experiment after the tumor volume reached at 200 sequence was cloned into the BamHI and BglII sites of the mm3. pDON-AI2 vector (Takara) and transfected into G3T-hi

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packaging cells. All the infected cells were cultured in a by staining with 5 mmol/L calcein-AM. Cell viability was medium containing the appropriate antibiotics. The control determined by using a microscope to count viable cells. The shRNA-expressing retrovirus vector was made using the same number of viable cells just after treatment with drugs (0 procedure, but with the following oligonucleotide sequence hour) was set at 100. 50-GCAAGCTGACCCTGAAGTTCAGAGCTCACCGAACTTCA- GGGTCAGCTTGC-30. Expression vectors for mouse ATF4 were Immunohistochemical analysis also constructed using cDNAs obtained from RT-PCR derived Theviablecellswerestainedwith5mmol/L calcein-AM. from mouse hepatic RNA. Coding regions were ligated into the After staining with calcein-AM, cells were fixedwith4%(w/ pcDNA3.1 (þ) vector (Invitrogen). v) paraformaldehyde in phosphate buffer, pH 6.9, and thereafter treated with 10% normal sheep serum for 1 hour. Cells Small interfering RNA were incubated with the rabbit anti-p53 antibodies (sc-6243; Oncogenic transformed Atf4 / cells were transfected with Santa Cruz Biotechnology) at 4C for 24 hours. After washing siRNA against p19Arf (Santa Cruz Biotechnology). This siRNA with cold PBS, cells were incubated with anti-rabbit IgG is designed to prevent the expression of p19ARF, and the Cy3-conjugated antibodies (Sigma) for 12 hours. The viable downregulation efficacy was confirmed in previous studies and p53 positive cells were detected under a 20 objective (19). On day 7 after oncogene introduction, cells were trans- lens. fected with siRNA (20 nmol/L) and used for experiments at 48 hours after transfection. Statistical analysis The statistical significance of differences among groups was Determination of the binding capacity of MDM2 to p53 determined by ANOVA and post hoc Bonferroni comparisons. protein A 5% probability was considered significant. Tumor masses formed by oncogenic wild-type or Atf4 / cells were removed from tumor-bearing mice at ZT2 and ZT14. Results The masses were homogenized in a lysis buffer as described ATF4 regulates the stability of p53 protein through the earlier. Equal amounts of cell lysates (1 mg protein) were ARF–MDM2 pathway precleared with 30 mL of protein G-Sepharose beads. After We showed previously that the protein levels of p53 were precleaning, 1 mg anti-p19ARF antibodies (sc-32748; Santa significantly higher in oncogenic Atf4 / cells than in normal Cruz Biotechnology) and 30 mL protein G-sepharose beads cells (19). To investigate the role of ATF4 in the expression of were added into lysates, and the mixtures were incubated for p53 in malignant tumor cells, the levels of p53 protein and its 12 hours at 4C. The beads and supernatants were separated by mRNA were assessed in ATF4-downregulated tumor cells. In centrifugation. The beads were washed 3 times with washing this experiment, UV.BAL-5.4G cells were used because of buffer and subjected to Western blot analysis. After addition of excessive ATF4 expression (data not shown). Infection of rGST-p53 protein into the supernatants (1 mg/mL), the mix- UV.BAL-5.4G with retrovirus vector-expressing shRNA target- tures were further immunoprecipitated with anti-MDM2 anti- ing Atf4 resulted in a dose-dependent inhibition of ATF4 bodies (sc-965; Santa Cruz Biotechnology) as described earlier. protein expression (Fig. 1A, top). Downregulation of ATF4 The cell lysates, supernatants, and immunoprecipitations were significantly increased the protein levels of p53, but had little analyzed by 5%, 10%, or 15% SDS-PAGE followed by immuno- effect on its mRNA levels (Fig. 1A, bottom), suggesting that blotting with antibodies against p53 (sc-6243), p19ARF (sc- ATF4 regulates protein levels of p53 in tumor cells without 22784), or MDM2 (sc-812). induction of its mRNA expression. To investigate the possibility that ATF4 regulates the expres- Determination of cytotoxicity sion of p53 protein at posttranscriptional level, we explored the Tumor masses formed by UV.BAL-5.4G or oncogenic- stability of p53 protein in ATF4-downregulated tumor cells. transformed wild-type and Atf4 / cells were removed from UV.BAL-5.4G cells were infected with Atf4 shRNA- or control tumor-bearing mice at ZT2 or ZT14. Single-cell suspensions shRNA-expressing retrovirus vectors. Then 50 mg/mL of CHX were prepared by standard methods (19). Cell suspensions was added to the media, and cell lysates were prepared at were mixed with 100 mmol/L nutlin-3, 500 mmol/L bleomy- selected time intervals. In control shRNA-infected cells, the cin, or 1 mmol/L dacarbazine, and thereafter seeded on 24- half-life of p53 protein was approximately 0.65 hours (Fig. 1B). well plate. At 2 hours after seeding, we confirmed attach- However, the half-life in Atf4 shRNA-infected cells was pro- ment of cells on the well bottom. The culture medium was longed about 9-fold (5.9 hours). A deficiency in ATF4 in replaced to drug-free medium, and cells were incubated for malignant tumor cells seems to induce the stabilization of indicated times. The dosage of drugs was selected based on p53 protein. previous reports (20–23). A significant antitumor effect of The stability of p53 protein is mainly regulated by its nutlin-3 is observed in mice when they were administered phosphorylation and interaction with specific ubiquitin ligases orally with the drug at the dosage of 200 mg/kg (20). Oral MDM2, which promote p53 protein ubiquitination and its administration of the same dosage of nutlin-3 into mice proteasomal degradation (11–13). Although p19ARF (also results in peak plasma concentration at 100 mmol/L (21). known as p14ARF in humans) also stabilizes p53 by seques- Similar reason was also applicable in both cases of bleomy- tering MDM2 (11), the levels of both p19ARF mRNA and its cin and dacarbazine (22, 23). The viable cells were detected protein were significantly increased in ATF4-downregulated

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Figure 1. ATF4 regulates the stability of p53 protein in tumor cells. A, the levels of p53 protein and mRNA in Atf4-downregulated UV.BAL-5.4G cells. The levels of protein and mRNA were assessed by Western blotting and real-time RT-PCR, respectively. Cells were infected with retrovirus vectors expressing Atf4 shRNA at indicated amounts (ng). The top panel shows the immunoblotting of ATF4, p53, and ACTIN proteins. B, inﬂuence of ATF4 downregulation on the stability of p53 protein in UV.BAL-5.4G cells. The half-lives of the p53 protein were calculated using the regression equation. C, inﬂuence of ATF4 downregulation on the levels of p19ARF and its mRNA in UV.BAL-5.4G cells. D, the protein levels of ATF4, p19ARF, MDM2, and p53 in oncogenic Atf4 / cells. E, suppressive action of ATF4 on the expression of p19ARF and p53 proteins in oncogenic Atf4/ cells. F, the effect of p19ARF downregulation on the protein levels of p53 in oncogenic Atf4/ cells. For intensity plots, the mean values of basal level are set at 1.0. In A, B, C, and F, each value represents the mean SEM (n ¼ 3–6). , P < 0.01; , P < 0.05 compared with the control group (0 ng).

UV.BAL-5.4G cells (P < 0.01; Fig. 1C). These results suggest that ATF4 is required for circadian accumulation of p53 p19ARF is involved in the regulation of p53 protein stability by protein in tumor cells ATF4. The expression of p53 protein in the thymuses of mice To further evaluate the role of p19ARF in the regulation of exhibits circadian oscillation (16). In this study, we also found the stability of p53 protein by ATF4, we assessed the protein a signiﬁcant circadian accumulation of p53 protein in UV.BAL- levels of p53 and p19ARF in oncogenic Atf4 / cells. MEFs were 5.4G tumor cells implanted in mice (P < 0.05; Fig. 2A, top). The prepared from embryos of wild-type or Atf4 / mice, and circadian accumulation of p53 protein was occurred without infected with retroviral vectors expressing H-rasV12 and rhythmic change in its mRNA expression (Fig. 2A, bottom). A SV40LT for oncogenic transformation (19). As compared to similar time-dependent accumulation of p53 protein was also wild-type oncogenic cells, the protein levels of both p53 and detected in cultured UV.BAL-5.4G cells whose circadian clocks p19ARF were obviously abundant in oncogenic Atf4 / cells were synchronized by DEX treatment (Fig. 2B), suggesting that (Fig. 1D). The elevated levels of both p53 and p19ARF proteins the oscillation in the expression of p53 protein is cell in Atf4 / oncogenic cells were decreased by overexpression autonomous. of ATF4 (Fig. 1E). Furthermore, the levels of p53 protein in ATF4 acts not only as a regulator of p53 protein levels, but Atf4 / oncogenic cells were also decreased by downregula- also as a component of the mammalian circadian clock (18). To tion of p19ARF (Fig. 1F). Taken together, these results suggest investigate whether ATF4 regulates the circadian accumula- that ATF4 affects the stability of p53 protein by repressing the tion of p53 protein, we assessed the temporal expression expression of p19ARF. proﬁles of p53 in tumor masses formed by oncogenic wild-

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Regulation of Circadian Degradation of p53 Protein by ATF4

Figure 2. Cell autonomous regulation of circadian accumulation of p53 protein in tumor cells. A, temporal expression profile of p53 protein (top) and its mRNA (bottom) in UV.BAL-5.4G tumor cells implanted in mice. For intensity plots, the mean value of ZT2 is set at 1.0. B, temporal expression profile of p53 (top) and its mRNA (bottom) in cultured UV.BAL-5.4G tumor cells after 10 nmol/L DEX treatment for 2 hours. For intensity plots, the basal value (0 hour) is set at 1.0. Each value represents the mean SEM (n ¼ 4– 6). There were significant time- dependent variations in p53 protein levels in both panels (P < 0.05 for A and B, respectively).

type or Atf4 / cells. As compared with wild-type tumor cells, ATF4 regulates the circadian change in the p53 protein the p53 protein levels in Atf4 / tumor cells increased at both stability through the p19ARF–MDM2 pathway ZT2 and ZT14, but the protein levels did not show a significant MDM2 protein in UV.BAL-5.4G tumor masses was constant- time-dependent variation (Fig. 3A). Because there was no ly expressed throughout the day, whereas the levels of both significant time-dependent oscillation of p53 mRNA levels in ATF4 and p19ARF proteins in the tumor masses exhibited both wild-type and Atf4 / tumor cells (Supplementary Fig. obvious circadian oscillations (Fig. 4A, top). The accumulation S2), ATF4 seems to regulate the circadian accumulation of p53 of p19ARF varied almost inversely with ATF4 protein expres- protein at posttranscriptional level. sion. The levels of p19Arf mRNA in UV.BAL-5.4G tumor masses To test this possibility, we explored whether ATF4 induces a showed a significant circadian oscillation, with higher levels circadian change in the stability of p53 protein in tumor cells. from the late light phase to the early dark phase (P < 0.05; Fig. As shown in Fig. 3A, the levels of p53 protein in wild-type tumor 4A, bottom). Similar significant time-dependent expression of masses prepared at ZT2 were low. We thus conducted immu- p19ARF was also detected in wild-type tumors (P < 0.05; Fig. noprecipitation by using 2 types of anti-p53 antibodies to 4B). However, neither p19ARF mRNA nor protein showed detect low levels of p53 protein. In the wild-type tumor cells, significant time-dependent oscillation in the Atf4 / cell the half-life of p53 proteins varied depending on the time of tumor masses. The protein levels of p19ARF was elevated at their preparation. The half-life of p53 protein in wild-type both time points, suggesting that constant elevation of p19ARF tumor cells prepared at ZT2 was much shorter than that of in Atf4 / cells may prevent the circadian change in the MDM2 p53 protein in cells prepared at ZT14 (Fig. 3B). In contrast, the binding to p53 protein. half-life of p53 protein in Atf4 / tumor cells was considerably MDM2 promotes the degradation of p53 protein by func- longer than that from wild-type cells, but the time of prepa- tioning as an E3 ubiquitin ligase. The ubiquitination occurs ration did not cause significant differences in the half-life of through binding of MDM2 to the transactivation domain of the p53 protein in Atf4 / tumor cells (Fig. 3B). These results p53 protein (11–13). To investigate the circadian time depen- suggest that circadian accumulation of p53 protein in wild- dency of the binding ability of MDM2 to p53 protein, we type tumor cells is associated with the time-dependent change conducted immunoprecipitation experiment as shown in its stability; the circadian change in that stability is caused by in Fig. 4C. The protein levels of p53 in wild-type tumor cells ATF4. prepared at ZT14 were approximately 4-fold higher than those

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Figure 3. ATF4 regulates circadian accumulation of p53 protein in tumor cells. A, temporal expression proﬁle of p53 protein in oncogenic wild-type and Atf4 / cells implanted in mice. For intensity plots, the mean value of ZT2 in the wild-type group is set at 1.0. Each value represents the mean SEM (n ¼ 6). , P < 0.05; comparison is between groups. B, the effect of ATF4 deﬁciency on the time-dependent stability of p53 protein. Single tumor cell suspensions were prepared at ZT2 or ZT14 from oncogenic wild-type or Atf4/ cells implanted in mice. Cells were incubated in the presence of 50 mg/mL cycloheximide (CHX), and nuclear fractions were extracted at indicated times. The immunoprecipitated p53 protein was detected by Western blotting. For intensity plots, the mean basal value (0 hour) of each group is set at 1.0. Each value represents the mean SEM (n ¼ 3). , P < 0.05; comparison is between groups.

at ZT2 (Fig. 3A). Such large difference in p53 contents could ATF4 forms heterodimers with C/EBPa that act to repress hinder accurate comparison of the binding capacity of MDM2 p19Arf gene transcription through the C/EBP-ATF response to p53 between the 2 times. We thus added constant amount of element (CARE; ref. 19). The CARE sequence is located between rGST-p53 protein in tumor cell lysates and evaluated the 6263 and 6255 with respect to the transcriptional start site binding capacity of MDM2 to exogenous p53 protein. The of the p19Arf gene. Chromatin immunoprecipitation analysis results of immunoprecipitation analysis revealed that MDM2 revealed that the amount of ATF4 binding to CARE within the protein in wild-type tumor cells precipitated together with p19Arf 50-ﬂanking region varied time dependently (Supple- p19ARF in a circadian time-dependent manner (Fig. 4C, IP: mentary Fig. S3). These results suggest that ATF4 periodically p19ARF). The amount of MDM2 associated with p19ARF was suppresses the expression of p19Arf mRNA, thereby causing greater at ZT14 compared with that at ZT2. Consequently, the circadian oscillation in its protein levels. much amount of p19ARF-unbound MDM2 remained in supernatants prepared from ZT2 of wild-type tumor cell lysates (Fig. Dosing time-dependent change in the cytotoxicity of 4C, Supernatant A). The p19ARF-unbound MDM2 in wild-type chemotherapeutic agents tumor cells precipitated together with rGST-p53 in a circadian Because the p53 protein levels in UV.BAL-5.4G and onco- time-dependent manner (Fig. 4C, IP: MDM2). The amount of genic wild-type tumor cells oscillated in a circadian time- rGST-p53 associated with MDM2 was greater at ZT2 compared dependent manner, we investigated whether that oscillation to that at ZT14. affects the cytotoxic effect of chemotheraputic agents. Several In Atf4 / tumor cells, most of p19ARF in Atf4 / tumor cell studies have showed that the dosing time-dependent change in lysates precipitated with anti-p19ARF antibody, but substan- the cytotoxicity of chemotheraputic agents is associated not tial amount of p19ARF-unbound MDM2 still remained in the only with circadian oscillation in the sensitivity of tumor cells supernatants (Fig. 4C, Supernatant A). The p19ARF-unbound to the drugs but also with their pharmacokinetics (24–25). To MDM2 in the supernatants prepared from Atf4 / cells could exclude the inﬂuence of circadian change in the distribution of be associated with rGST-p53, but the association between drugs into the tumor cells, single-cell suspensions were MDM2 and rGST-p53 did not exhibit the time-dependent prepared from UV.BAL-5.4G tumor masses at ZT2 and ZT14 variation (Fig. 4C, IP: MDM2). These results suggest that the (Fig. 5A). Cells were treated with chemotheraputic agents, and time-dependent interaction between p19ARF and MDM2 p53 protein levels and cell viability were determined at indi- underlies circadian change in the binding capacity of MDM2 cated times. to p53 protein. Constant interaction between p19ARF and At 8 hours after treatment with nutlin-3, a potent inhibitor MDM2 in Atf4 / tumor cells may prevent the circadian of p53 degradation, the number of p53-positive cells and oscillation of MDM2 binding to p53 protein. their signal intensity were obviously increased, but the

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Figure 4. ATF4 regulates the circadian change in the stability of p53 protein through p19ARF-MDM2 pathway. A, temporal expression profile of ATF4, p19ARF, MDM2, ACTIN proteins (top), and p19Arf mRNA (bottom) in UV.BAL-5.4G cells implanted in mice. For intensity plots, the mean value of ZT2 was set at 1.0. Each value represents the mean SEM (n ¼ 4–6). There was a significant time-dependent variation in p19Arf mRNA levels (P < 0.05, ANOVA). B, temporal expression profile of ATF4, p19ARF, MDM2, b-ACTIN proteins (top), and p19Arf mRNA (bottom) in oncogenic wild- type and Atf4 / cells implanted in mice. For intensity plots, the mean value of ZT2 in the wild-type group is set at 1.0. Each value represents the mean SEM (n ¼ 6). , P < 0.01; , P < 0.05; comparison is between groups. C, influence of ATF4 deficiency on the time-dependent interaction between p19ARF and MDM2 in tumor cells. Left panel shows experimental procedure of immunoprecipitation analysis. Tumor cell lysates were prepared from oncogenic wild-type and Atf4/ cells implanted in mice at ZT2 or ZT14. Immunoprecipitation was conducted using antibodies against p19ARF and MDM2. The protein levels of p19ARF, MDM2, and rGST-p53 in whole cell lysates, supernatant, and immunoprecipitation (IP) complexes were assessed by Western blotting (WB).

accumulation of p53 protein was greater when cells were was also found in tumor cells treated with genotoxic drugs, treated with nutlin-3 at ZT14 (Fig. 5B). Consistent with the bleomycin and dacarbazine (Fig. 5D, middle and right panels). dosing time dependency of p53 accumulation, induction in the The cytotoxic effect of these drugs was enhanced at the time of expression of p53 target genes, Bax, p21, Noxa, and Puma was day when p53 protein is abundant. also significantly enhanced in cells treated with nutlin-3 at In the final set of experiment, we investigated whether ATF4 ZT14 (P < 0.05, respectively; Fig. 5C). These findings suggest was involved in the dosing time-dependent change in the cyto- that the growth-suppressive properties of p53 are changed toxic effect of nutlin-3. To this end, cell viability was assessed in according to the dosing time of nutlin-3. In fact, the cytotoxic nutlin-3–treated wild-type and Atf4 / tumor cells. Significant effect of nutlin-3 on UV.BAL-5.4G tumor cells also varied dosing time-dependent difference in nutlin-30s cytotoxic effect significantly depending on the time when the cells were treated was found when the drug was applied to cells prepared from (Fig. 5D, left). The effect in cells prepared at ZT 14 was greater wild-type tumor masses (P < 0.01; Fig. 6). Conversely, there was than in cells prepared at ZT2. Similar dosing time dependency no significant dosing time-dependent difference in the

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Figure 5. Circadian change in the sensitivity of tumor cells to chemotherapeutic agents. A, schematic experimental procedure for evaluating the dosing time dependency of cytotoxic effects of nutlin-3 or genotoxic drugs on tumor cells. Single tumor cell suspensions were prepared at ZT2 or ZT14 from mice implanted with tumor cells. Cells were seeded on 24-well plate, and immediately incubated in the presence or absence of each drug for 2 hours. B, dosing time dependency of the nutlin-3–induced accumulation of p53 protein in UV.BAL-5.4G tumor cells. Double-labeled cells (either yellow or red nucleus surrounded by green cytoplasm) indicate the p53-positive cells. C, dosing time dependency of the nutlin-3–induced expression of p53 target genes in UV.BAL-5.4G tumor cells. The levels of p53 target genes were assessed at indicated times by RT-PCR. For intensity plot, the mean basal value (0 hour) of ZT2 group is set at 1.0. , P < 0.05 is compared with ZT2 groups. #, P < 0.05 is compared with basal levels (0 hour). D, dosing time dependency of the cytotoxic effect of nutlin-3, bleomycin, and dacarbazine on UV.BAL-5.4G cells. For plots of cell survival rate, the mean basal value (0 hour) of each group is set at 100. Each value represents the mean SEM (n ¼ 3–5) , P < 0.01; , P < 0.05; comparison is between groups.

cytotoxic effect of nutlin-3 on oncogenic Atf4 / cells. Fur- intensity in nutlin-3–treated Atf4 / cells were greater than thermore, the viability of nutlin-3–treated Atf4 / cells was those in wild-type cells (Fig. 6), a potent accumulation of p53 more rapidly decreased as compared with wild-type protein may induce the severe cytotoxic effect of nutlin-3 on cells. Because the number of p53-positive cells and their signal Atf4 / tumor cells at both time points.

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Regulation of Circadian Degradation of p53 Protein by ATF4

Figure 6. ATF4 is required for circadian change in the sensitivity of tumor cells to nutlin-3. Single tumor cell suspensions were prepared at ZT2 or ZT14 from oncogenic wild- type or Atf4 / cells implanted in mice. Cells were treated with 100 mmol/L nutlin-3 as shown in Fig. 5A. For plots of cell survival rate, the mean basal value (0 hour) of each group is set at 100. Each value represents the mean SEM (n ¼ 3). , P < 0.01; , P < 0.05; comparison is between groups. The microscopic photographs show the temporal changes in the nutlin-3–induced accumulation p53 protein in oncogenic wild-type (top) and Atf4/ cells (bottom). Double- labeled cells (either yellow or red nucleus surrounded by green cytoplasm) indicate the p53-positive cells.

Discussion during those times of day when p19ARF–MDM2 interaction The molecular circadian clock operates at a cellular level and was facilitated (Fig. 7). In contrast, the p53 mRNA levels in governs a wide variety of physiologic processes. This study tumor cells failed to exhibit a significant circadian oscillation, suggests that this clockwork governs the time-dependent suggesting that the molecular clock regulates the circadian accumulation of p53 protein in tumor cells. In mammals, expression of p53 protein at posttranscriptional level. In fact, circadian rhythmicity in various biological processes is con- the abundance of p53 protein in Atf4 / tumor cells increased trolled by a molecular pacemaker composed of clock gene consistently throughout the day without a corresponding products (26–28). These gene products constitute an oscillat- increase in mRNA levels. ing mechanism based on self-sustained transcriptional/trans- In this study, we used retrovirus vectors expressing SV40LT lational feedback loops. The Clock gene encodes the CLOCK as a transforming agent, together with H-rasV12. Because transcription factor, which dimerizes with BMAL1 to activate SV40LT inactivates p53 and prevents the phosphorylation of the transcription of Period (Per) and Cryptochrome (Cry) genes Rb by protein–protein interaction (32, 33), inactivation of the via E-box or E-box-like enhancer sequences (4, 29). Once PER growth-suppressive properties of p53 has been shown to be and CRY proteins have reached critical concentrations, they essential for immortalization of MEFs by SV40LT. In fact, p53 attenuate CLOCK/BMAL1 transactivation, thereby generating protein in oncogene-introduced wild-type cells was precipi- circadian oscillation in their own transcription. In addition, tated together with SV40LT (Supplementary Fig. S4). However, CLOCK/BMAL1, PER, and CRY proteins regulate the expres- substantial amount of SV40LT-unbound p53 protein still sion of ATF4, which in turn sustains the transcriptional existed in wild-type cells, suggesting that extensive expression oscillation of Bmal1, Per2, and Cry1 gene (18). This mechanism of p53 in oncogene-introduced cells overrides the binding interconnects the positive and negative limbs of circadian capacity of SV40LT. Concomitant introduced H-rasV1 may also clockwork circuitry and also regulates 24-hour variation in enhance the accumulation of p53 protein through DNA dam- output physiology through the periodic activation/repression age response. The SV40LT-unbound p53 protein in wild-type of clock-controlled output genes (30). ATF4 also acts as an cells seemed to be involved in the circadian regulation of tumor output component of the circadian clock and regulates the cell sensitivity to chemotherapeutic agents. rhythmic expression of its target genes through the CRE (31). Because cells deficient in p53 exhibit resistance to chemo- In murine fibroblast tumor cells, ATF4 functioned as a therapeutic agents (34, 35), we explored whether the oscillation circadian regulator of p19ARF expression, causing a time- of p53 protein levels affects the susceptibility of tumor cells to dependent interaction between p19ARF and MDM2, which chemotherapeutic agents. Nutlin is a cis-imidazoline analog resulted in periodic prevention of MDM2-mediated p53 deg- that mimics 3 residues (Phe19, Trp23, and Leu26) in the helical radation. Consequently, the half-life of p53 protein varied in a region of p530s transactivation domain, residues that are circadian time-dependent manner; the proteins accumulated conserved across species and that are critical for binding to

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Horiguchi et al.

conventional chemotherapeutic agents, bleomycin and dec- arbazine, were also enhanced by applying the drugs at the times of day during which accumulation of p53 protein was facilitated in tumor cells. Because there was no signiﬁcant dosing time-dependent difference in the cytotoxic effect of nutlin-3 on oncogenic Atf4 / cells, ATF4-mediated regulation of the ARF–MDM2 pathway could cause a circadian accumulation of p53 protein, thereby causing the time-dependent change in the susceptibility to chemotherapeutic agents. The amount of p53 protein in oncogene-introduced Atf4 / cells not only failed to show circadian variation, but also was greater than that in wild-type cells. This may be because of a decrease in the degradation of p53 through MDM2-mediated proteasomal pathway. Although the protein levels of p19ARF were elevated in Atf4 / cells, MDM2 was not completely entrapped by p19ARF. The p19ARF-unbound MDM2 in Atf4 / cells still had a capacity to bind to p53 protein. Nutlin-3 seemed to prevent the interaction between p19ARF-unbound MDM2 and p53 protein, resulting in severe cytotoxic effect on Atf4 / cells. The effectiveness and toxicity of chemotherapeutic agents vary with the dosing time. However, many drugs are still administered without regard to the time of day. Identiﬁcation of the mechanism underlying the dosing time dependency of chemotherapeutic agents should help achieve better chrono- pharmacotherapy for treatment of cancers.

Disclosure of Potential Conﬂicts of Interest No potential conﬂicts of interest were disclosed.

Figure 7. Schematic mechanism of the circadian accumulation of p53 protein in tumor cells. ATF4 induced the circadian expression of p19ARF; Authors' Contributions Conception and design: M. Horiguchi, S. Koyanagi, S. Ohdo the oscillation of p19ARF protein interacted time-dependently with Development of methodology: M. Horiguchi, S. Koyanagi, N. Matsunaga MDM2, resulting in periodic repression of MDM2-mediated ubiquitylation Acquisition of data (provided animals, acquired and managed and degradation of p53 protein. Consequently, p53 proteins accumulate patients, provided facilities, etc.): M. Horiguchi, S. Koyanagi, K. Kakimoto, during those times of day when p19ARF-MDM2 interaction was N. Matsunaga facilitated. The circadian accumulation of p53 protein seems to cause a Analysis and interpretation of data (e.g., statistical analysis, biostatistics, time-dependent increase in the sensitivity of cancer cells to computational analysis): M. Horiguchi, S. Koyanagi, N. Matsunaga, C. Yama- chemotherapeutic agents. shita, S. Ohdo Writing, review, and/or revision of the manuscript: M. Horiguchi, S. Koyanagi, S. Ohdo MDM2. Nutlin binds to MDM2 in the p53-binding pocket and Administrative, technical, or material support (i.e., reporting or orga- stabilizes the p53 protein (20). The stabilization of p53 leads to nizing data, constructing databases): A. M. Hamdan Study supervision: M. Horiguchi, S. Koyanagi, C. Yamashita, S. Ohdo cell-cycle arrest, apoptosis, and growth inhibition of tumor cells including myeloma, acute myeloid leukemia, acute lym- Acknowledgments phoblastic leukemia, and B-cell chronic lymphocytic leukemia We are grateful for technical support from the Research Support Center, (36). Nutlin-3 also exhibited a significant cytotoxic effect on Graduate School of Medical Sciences, Kyushu University. Our thanks to Mr. Steven Sabotta for proofreading the article. murine fibroblast tumor cells, but the effect on wild-type cells varied according to its dosing time. The cytotoxic effect of Grant Support nutlin-3 on wild-type cells was further enhanced by applying This study was partially supported by a Grant-in-Aid for Scientific Research the drug to cells prepared at the early dark phase (ZT14). (B) (S. Ohdo, 21390047 and S. Koyanagi, 24390149) from the Japan Society for the Promotion of Science. During this circadian phase, p53 protein was more accumu- The costs of publication of this article were defrayed in part by the payment lated after nutlin-3 treatment. The dosing time-dependent of page charges. This article must therefore be hereby marked advertisement difference in nutlin-3–induced p53 accumulation seems to in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. cause a circadian change in the sensitivity of cancer cells to Received July 3, 2012; revised January 28, 2013; accepted February 18, 2013; chemotherapeutic agents. In fact, the cytotoxic effects of published OnlineFirst April 10, 2013.

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Rhythmic Control of the ARF-MDM2 Pathway by ATF4 Underlies Circadian Accumulation of p53 in Malignant Cells

Michiko Horiguchi, Satoru Koyanagi, Ahmed M. Hamdan, et al.

Cancer Res 2013;73:2639-2649. Published OnlineFirst April 11, 2013.

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