DNA Damage–Specific Control of Cell Death by Cryptochrome in P53

Published OnlineFirst November 13, 2012; DOI: 10.1158/0008-5472.CAN-12-1994

Cancer Therapeutics, Targets, and Chemical Biology Research

DNA Damage–Speciﬁc Control of Cell Death by Cryptochrome in p53-Mutant Ras–Transformed Cells

Jin Hyup Lee, Shobhan Gaddameedhi, Nuri Ozturk, Rui Ye, and Aziz Sancar

Abstract The main feedback loop driving circadian rhythm in mice is controlled, in part, by the genes encoding the cryptochromes Cry1 and Cry2. Targeted mutation of both Cry1 and Cry2 delay the early onset of tumor formation in p53-null mutant mice. Furthermore, Ras-transformed p53- and Cry-null mouse skin ﬁbroblasts are more sensitive than p53 mutants to apoptotic cell death initiated by agents that activate either the intrinsic or the extrinsic apoptosis pathways. Here, we investigated the effect of Cry1 and Cry2 mutations on cell death by other genotoxic agents that generate alkylated bases, interstrand crosslinks, DNA–protein crosslinks, and double- strand breaks. Both ultraviolet (UV) and the UV mimetic compound oxaliplatin and the radiomimetic compound doxorubicin promoted apoptosis by upregulating the tumor suppressor p73. However, only the UV and oxaliplatin-induced upregulation of p73 mediated by the transcription factor Egr1, but not the doxorubicin- induced upregulation mediated by the transcription factor E2F1, was enhanced by Cry1/Cry2 double mutation. Accordingly, Egr1 downregulation reduced oxaliplatin-induced apoptosis, whereas E2F1 downregulation reduced doxorubicin-induced apoptosis. Our ﬁndings establish distinct roles for cryptochromes in intrinsic apoptosis induced by UV mimetic and radiomimetic agents. Cancer Res; 73(2); 785–91. 2012 AACR.

Introduction its nuclear export enabling the transactivators to upregulate Cryptochrome (Cry) is the main repressor in the transcrip- p73 transcription (4). Interestingly, of these 2 transcriptional tion-translation feedback loop that generates circadian rhyth- activators, Egr1 is a circadian clock controlled gene with a micity in mice (1). Recently, we reported that Cry mutation in robust rhythmicity (9), whereas E2F1 is not controlled by the Ras-transformed p53-null mouse skin fibroblasts sensitized circadian clock (10). While investigating the effect of circadian the cells to apoptosis by ultraviolet (UV) and UV mimetic clock disruption on cellular response to DNA damage, we agents such as oxaliplatin (2, 3). Further analysis revealed that discovered that circadian clock disruption by Cry mutation this enhanced apoptosis was caused by the amplified upregu- led to elevated level of expression of Egr1, which resulted in lation of the p73 tumor suppressor due to the high level of the high level of expression of p73 upon DNA damage by UV or clock controlled transcription factor Egr1 in Cry-null cells (3). oxaliplatin and enhanced apoptosis resulting in increased As p73 is a DNA damage–inducible gene (4, 5), we wished to clonogenic cell death by these genotoxicants (3). find out the effect of other DNA-damaging agents on p73 Here, we wished to investigate other DNA-damaging agents induction and apoptosis in cells with and without a functional that generate DNA lesions that encompass the entire spectrum circadian clock. The p73 gene has a complex regulatory mech- of repair pathways for their effects on p73-medaited apoptosis fi anism that includes E2F1, Egr1, and C-EBPa transcription in the absence or presence of Cry in p53-null cells to nd out factors. There are 1 C-EBPa-, 3 E2F1-, and 5 Egr1-binding sites whether Cry mutation sensitizes these cells to all genotoxi- in the p73 promoter (4–8). The p73 protein level is mainly cants. We used UV and oxaliplatin (UV mimetic) whose major regulated by transcription that is activated by E2F1 and Egr1 lesions are repaired by nucleotide excision repair, ethyl- and is repressed by C-EBPa. DNA damage by doxorubicin or methane sulfonate (EMS) that induces base alkylation repaired cisplatin causes dissociation of C-EBPa from the promoter and by base excision repair, doxorubicin that induces double- strand breaks repaired by nonhomologous end-joining and homologous recombination, mitomycin C that induces interstrand crosslinks repaired by recombination and translesion Authors' Affiliation: Department of Biochemistry and Biophysics, Univer- – sity of North Carolina, School of Medicine, Chapel Hill, North Carolina synthesis, and camptothecin that induces DNA protein crosslinks repaired by proteolysis and recombination (11, 12). We Note: Supplementary data for this article are available at Cancer Research Online (http://cancerres.aacrjournals.org/). found that at equitoxic doses, only the UV mimetic (UV and oxaliplatin) and the radiomimetic (doxorubin and camptothe- Corresponding Author: Aziz Sancar, University of North Carolina, Chapel Hill, Campus Box 7260, 3073 Genetic Medicine, Chapel cin) agents induced p73 expression. Furthermore, surprisingly, Hill, NC 27599. Phone: 919-962-0115; Fax: 919-966-2852; E-mail: we found that while oxaliplatin induces p73 through Egr1- [email protected] mediated transcriptional upregulation, doxorubicin induces doi: 10.1158/0008-5472.CAN-12-1994 p73 through E2F1-mediated transcriptional upregulation. As a 2012 American Association for Cancer Research. consequence the clonogenic killing and apoptotic effects of

www.aacrjournals.org 785

Lee et al.

oxaliplatin are enhanced by Cry mutation that causes consti- antibodies produced in our laboratory have been described tutive upregulation of Egr1, but the cell killing and apoptotic previously (2, 3). For immunoblot analysis, cells were lysed in a effects of doxorubicin are not affected by Cry mutation. As radioimmunoprecipitation assay (RIPA) buffer (50 mmol/L oxaliplatin plus doxorubicin combination is used in various Tris-HCl, pH 7.6, 1% Nonidet P-40, 140 mmol/L NaCl, 0.1% chemotherapeutic regimens (13), these ﬁndings may help in SDS) supplemented with complete protease inhibitor cocktail designing more effective drug delivery protocols for cancer (Roche), and 50 mg of lysate were loaded to precast 4% to 12% therapy. gradient SDS-PAGE (Invitrogen).

Immunoprecipitation Materials and Methods Cells were lysed in RIPA buffer supplemented with com- Establishment of p53 / and p53 / Cry1/2 / cell lines plete protease inhibitor cocktail (Roche). After preclearing Wild-type and p53 / mice of C57BL/6J background were with protein A/G agarose plus (Santa Cruz) for 1 hour at 4C, obtained from The Jackson Laboratory and p53 / Cry1/2 / whole-cell lysates were used for immunoprecipitation with mice in C57BL/6J background were generated in our labora- the indicated antibody. One microgram of the commercial tory (2). These cell lines were authenticated by microarray antibody was added to 1 mL of the cell lysate and incubated analysis using the whole genome oligo microarrays (Agilent at 4C for 8 to 12 hours. After adding protein A/G agarose Technologies) for mouse-specific gene expression analysis. beads, incubation was continued for an additional 1 hour. The Ras-transformed p53 / and p53 / Cry1/2 / cell lines The immunoprecipitates were then washed extensively with were generated in our laboratory as described previously RIPA buffer and eluted by boiling them with an SDS loading (3). These cells were maintained in Dulbecco's Modified buffer for 5 minutes. Eagle's Medium (DMEM) supplemented with penicillin, strep- tomycin, and 10% FBS. The cells were maintained in an Statistical analysis incubator at 37 C under 5% CO2. Values are shown as mean SD of at least 3 experiments calculated using the 2-tailed Student t test. Chemicals and siRNA Oxaliplatin, doxorubicin, camptothecin, mitomycin C, and Results EMS were purchased from Sigma. DharmaFECT (Dharmacon) Clonogenic killing by various genotoxicants of p53 / reagent was used according to the manufacturer's instruction and p53 / Cry1/2 / cells for transfection of ON-TARGET plus SMARTpool or single We used genotoxic treatments that generate DNA lesions siRNA duplexes obtained from Dharmacon [Egr1 (L-040286- repaired by all of the major repair pathways (base excision, 00-0005, J-040286-08-005, J-040286-07-005), E2F1 (L-044993-00- nucleotide excision, double-strand break repair, interstrand 0005, J-044993-16-0005, J-044993-15-0005) or nontargeting crosslink repair, DNA–protein crosslink repair) to examine the fi siRNA (D-001810-10-05) as a control]. To ensure speci city effects of clock disruption by Cry mutation on these repair of Egr1 and E2F1 downregulation by pooled siRNAs, we also pathways. Figure 1 shows the result of clonogenic assays for all conducted downregulation by 2 single siRNAs targeting the these treatments. In agreement with our previous report, the relevant genes and obtained essentially the same results. killing by UV (Fig. 1A) and oxaliplatin (UV mimetic agent, The effects of downregulation of Egr1 and E2F1 by siRNA are Fig. 1B), which produce bulky DNA adducts that are repaired fi speci c because downregulation of one does not affect the level exclusively by nucleotide excision repair (11), is enhanced by of the other transcription factor (Supplementary Fig. S1). Cry mutation. Interestingly, the killing effect of none of the other DNA damaging agents is influenced by Cry (Fig. 1C–F). Clonogenic cell survival assay This suggests that Cry mutation specifically affects the letha- The clonogenic cell survival assay was done as described lity of UV mimetic agents that produce bulky DNA adducts. fi previously with some modi cations (2). Cells were seeded at We have previously shown that Cry mutation enhanced low density to ensure the formation of 200 colonies per 6-well the clonogenic cell death of p53-null cells by UV and oxali- plate. Following plating, cells were kept in growth medium platin by amplifying the intrinsic apoptotic response to these for 10 to 14 hours and treated with indicated ranges of UV agents through increased transcription of p73 tumor sup- 2 given at a dose rate of 0.32 J/m /s or incubated with indicated pressor (3). Therefore, we decided to analyze the response concentrations of chemicals for the duration of the experi- of p53 / and p53 / Cry1/2 / cells to these various DNA- > ment. Readily visible colonies ( 50 cells per colony) formed damaging agents to find out whether the apoptosis enhance- after a 9- to 10-day incubation were stained with 5% methylene ment by Cry mutation was restricted to apoptosis induced by blue and counted. UV mimetic agents.

Protein analysis The following commercial antibodies and reagents were Effect of Cry mutation on apoptosis induced by full used: E2F1 and Actin (Santa Cruz Biotechnology); cleaved- spectrum of genotoxicants caspase3, cleaved-PARP1, and Egr1 (Cell Signaling Technolo- We conducted apoptosis assays at equitoxic doses of all 6 gy); p73 (Upstate Biotechnology); acetyl-lysine (Millipore); genotoxicants used in the survival assay by using doses that phospho-E2F1 (Rockland); and IgM (Thermo Scientiﬁc). Cry1 gave 50% survival in the clonogenic assay. We probed for

786 Cancer Res; 73(2) January 15, 2013 Cancer Research

Cryptochrome Mutation and DNA Damage–Induced Apoptosis

A B C

100 p53-/- 100 100 p53-/-Cry1/2-/- 80 80 80

60 60 60

40 40 40 Viability (%) Viability (%) Viability (%)

20 20 20

0 0 0 0 2 4 6 8 10 0 4 8 12 16 20 0 2 4 6 8 10 UV (J/m2 ) Oxaliplatin (µmol/L) Doxorubicin (µmol/L) D E F

100 100 100

80 80 80

60 60 60

40 40 40 Viability (%) Viability (%) Viability (%)

20 20 20

0 0 0 0 0.4 0.8 1.2 1.6 2 0 1 2 3 4 5 0 1 2 3 4 5 Camptothecin (µmol/L) Mitomycin C (µmol/L) EMS (mmol/L)

Figure 1. Effect of cryptochrome on clonogenic cell death by various genoxoticants. Cells of the indicated genotypes were irradiated or treated with the indicated dose of UV (A), oxaliplatin (B), doxorubicin (C), camptothecin (D), mitomycin C (E), or EMS (F) and then incubated for 9 to 10 days until colonies were readily visible. Colonies were stained with 5% methylene blue and then counted to obtain the UV survival curves. Results represent the means of 3 independent experiments (SD). apoptosis by measuring PARP and caspase-3 cleavage (14) response by upregulating p73 (3), we examined the effect of over a period of 12 hours from the start of UV irradiation or doxorubicin and UV/oxaliplatin along with those of other drug treatment. The results are shown in Fig. 2. Several points genotoxicants on p73 induction. emerge from this figure. First, even though equitoxic doses were used for all genotoxicants the extent of apoptosis at Mechanisms of p73 upregulation by radiomimetic and 12 hours in p53 / cells quite different for the various agents. UV mimetic agents It is remarkably high in doxorubicin-treated cells and negligi- We treated cells with the 6 genotoxic agents used in the ble in EMS-treated cells (Fig. 2A–D), indicating that, dependent clonogenic survival assay and followed p73 induction for on the genotoxicant, different apoptosis kinetics or other 12 hours. The results are shown in Fig. 3A. In agreement with modes of cell death are responsible for clonogenic killing. the apoptosis data, UV mimetic treatments (UV and oxalipla- Second, and most importantly, within the examined time tin)-caused p73 transcriptional upregulation in both p53 / þ þ frame, apoptosis is enhanced by Cry mutation only in (3) and p53 / (Supplementary Fig. S2) background is ampli- cells treated with UV and oxaliplatin, in agreement with the fied by Cry mutation. In contrast, the extent of p73 induction clonogenic survival data that show that Cry mutation enhances by doxorubicin and camptothecin is comparable to that only the killing effects of these 2 agents (Fig. 1A and B). Finally, achieved by UV mimetic treatments but is independent of the even though under our experimental conditions, the doxoru- Cry status of the cell line (Fig. 3A and B). Thus, it appears that bicin-induced apoptosis is not amplified by Cry mutation while UV/oxaliplatin and doxorubicin/camptothecin induce the extent of apoptosis induced by doxorubicin in p53 / cells p73 efficiently, there is a difference between the induction is comparable to that seen in p53 / Cry1/2 / cells treated mechanisms because one is affected by the status of Cry and with UV or oxaliplatin. In conclusion, the survival data com- the other is not. bined with the apoptosis data indicate that UV, oxaliplatin, To find out the cause of this differential effect of Cry on camptothecin, and doxorubicin induce apoptosis-associated cellular response to the 2 types of DNA lesions (bulky adducts cell killing in the absence of p53. Because we had previously vs. double-strand breaks) known to induce p73, we examined determined that the apoptosis induced in p53 / cells was the effects of these 2 types of DNA-damaging agents on mostly mediated by p73 and that Cry mutation enhanced this 2 transcription factors known to be involved in DNA

www.aacrjournals.org Cancer Res; 73(2) January 15, 2013 787

Lee et al.

A p53-/- p53-/-Cry1/2-/- B P < 0.005 0 3 6 12 0 3 6 12 h After treatment 0.4 0 h P < 0.01 UV 3 h 0.3 6 h Oxaliplatin 12 h 0.2 Doxorubicin c-PARP Camptothecin 0.1 Relative c-PARP levels Relative c-PARP levels

Mitomycin C 0.0

EMS

Actin

C -/- -/- -/- D p53 p53 Cry1/2 P < 0.01 0.4 0 3 6 12 0 3 6 12 h After treatment P < 0.01 0 h UV 3 h 0.3 6 h Oxaliplatin 12 h 0.2 Doxorubicin C casp-3 0.1 Camptothecin Relative C casp-3 levels levels Relative C casp-3

Mitomycin C 0.0

EMS

Actin

Figure 2. Effect of cryptochrome on apoptosis induced by various genotoxic agents. Cells were irradiated or treated with 5 Jm 2 UV, 10 mmol/L oxaliplatin 5 mmol/L doxorubicin, 1 mmol/L camptothecin, 2.5 mmol/L mitomycin C, or 2.5 mmol/L EMS for the indicated times. Cell lysates were probed for cleaved PARP (c-PARP; A) and cleaved caspase-3 (c-Casp3; C) by immunoblotting. Actin served as a loading control. Actin for EMS-treated cells was shown as a representative image. Quantiﬁcations of the levels of c-PARP (B) and c-Casp3 (D) normalized to actin are shown. The data are the mean of 3 independent experiments SD.

damage–induced upregulation of p73, the E2F1 and Egr1 clonogenic and apoptosis assays that the clock does not affect transcription factors (refs. 4–8; camptothecin behaves quite cellular response to double-strand breaks as determined by similarly to doxorubicin but as both act by similar mechan- these 2 endpoints. isms, double-strand break for doxorubicin, and double-strand We investigated the effects of E2F1 and Egr1 in more detail break associated with DNA–protein crosslink for campto- using doxorubicin (double-strand breaks) and oxaliplatin thecin, we chose to pursue only one in further experiments). (bulky adducts) as models for clock effects on cellular response The results are shown in Fig. 4. As seen in Fig. 4A, both UV and to the 2 types of DNA damage and also because these 2 drugs doxorubicin fail to induce Egr1 in the p53 / genetic back- are components of several frontline cancer therapy regimens. ground and Egr1 is constitutively elevated in p53 / Cry1/2 / The ﬁndings in Fig. 4A provide a plausible explanation for the cells regardless of DNA damage by either genotoxicants. differential effects of Cry mutation on cellular response to Importantly, however, doxorubicin upregulates E2F1 and damage by UV mimetic and radiomimetic agents: The upre- causes its phosphorylation and acetylation. These are neces- gulation of p73 by E2F1 following DNA damage by radio- sary for E2F1 activity but UV has no effect on E2F1 levels and mimetics is the predominant regulatory mechanism (15–18) posttranslational modiﬁcation (refs. 15–18; Fig. 4A). Of special and hence the elevated Egr1 does not contribute to doxoru- relevance, the effects of doxorubicin on E2F1 are independent bicin-induced p73 upregulation. In contrast, it appears that of the Cry status of the p53 / cell lines, consistent with the with UV mimetic treatment the Egr1-mediated transcription is

788 Cancer Res; 73(2) January 15, 2013 Cancer Research

Cryptochrome Mutation and DNA Damage–Induced Apoptosis

A B P < 0.005 p53-/- p53-/-Cry1/2-/- 0.4 0 h 0 3 6 12 0 3 6 12 h After treatment P < 0.005 3 h 0.3 UV 6 h 12 h Oxaliplatin 0.2

Doxorubicin 0.1

p73 Relative p73 levels Camptothecin 0.0 Mitomycin C

EMS

Actin

Figure 3. Differential p73 induction by cryptochrome in p53-null background. A, cells were irradiated with or exposed to 5 Jm 2 UV, 10 mmol/L oxaliplatin, 5 mmol/L doxorubicin, 1 mmol/L camptothecin, 2.5 mmol/L mitomycin C, or 2.5 mmol/L EMS for the indicated times, after which, cell lysates were analyzed by immunoblotting for p73. B, results of p73 quantiﬁcation are normalized to the expression of actin and shown. Actin for EMS-treated cells is shown as a representative image. Error bars indicate 1 SD from the mean (n ¼ 3).

the dominant mechanism, and as Egr1 (but not E2F1) is a enhanced apoptosis in p53 / Cry1/2 / cells compared with clock-controlled protein, in p53 / Cry1/2 / cells, the elevat- p53 / cells. The enhanced apoptosis is due to the upregula- ed Egr1 leads to enhanced p73 induction by UV mimetics and tion of p73 because knockdown of p73 by siRNA drastically

B p53-/- p53-/-Cry1/2-/- C p53-/- p53-/-Cry1/2-/- A Ctrl Egr1 Ctrl Egr1 :siRNA Ctrl Egr1 Ctrl Egr1 :siRNA - + - + - + - + Oxaliplatin - + - + - + - + Doxorubicin UV Doxorubicin c-PARP c-PARP

C casp-3 C casp-3

- + - + - + - + p73 p73 p-E2F1 Egr1 Egr1 E2F1 Actin Actin Egr1 D E p53-/- p53-/-Cry1/2-/- p53-/- p53-/-Cry1/2-/- Actin Ctrl E2F1 Ctrl E2F1 :siRNA Ctrl E2F1 Ctrl E2F1 :siRNA - + - + - + - + Oxaliplatin - + - + - + - + Doxorubicin Ace-Lyn IP: E2F1 IB c-PARP c-PARP E2F1 C casp-3 C casp-3

p73 p73

E2F1 E2F1

Actin Actin

Figure 4. Differential effect of cryptochrome on the p73-mediated apoptosis. A, cells were irradiated with 5 Jm 2 UV or treated with 5 mmol/L doxorubicin. Cell lysates were probed for Egr1 or total and phosphorylated forms of E2F1. Actin was used as a loading control. To probe acetylated forms of E2F1, cell lysates were immunoprecipitated with anti-E2F1 antibody. IB, immunoblotting; IP, immunoprecipitation. The immunoprecipitates were subjected to immunoblotting with anti-E2F1 and with antibody against acetylated lysines. B–E, effect of cryptochrome on the 2 types of p73 induction. Mouse ﬁbroblasts of the indicated genotypes were transfected with Egr1 (B and C) or E2F1 (D and E) siRNA, treated with 10 mmol/L oxaliplatin (B and D) or 5 mmol/L doxorubicin (C and E) and then analyzed for p73 expression directly by immunoblotting, and for apoptosis by immunoblotting for cleaved PARP and caspase-3.

www.aacrjournals.org Cancer Res; 73(2) January 15, 2013 789

Lee et al.

reduced apoptosis in both p53 / and p53 / Cry1/2 / cells. contribution of Cry to clonogenic cell death and to intrinsic (Supplementary Fig. S3). To further test this model, we down- apoptosis induced by a set of genotoxicants that generate regulated either Egr1 or E2F1 in p53 / and p53 / Cry1/2 / DNA lesions that are repaired by each of the major DNA backgrounds and tested the cell lines for p73 induction and repair pathways including base excision repair, nucleotide apoptosis. Figure 4B and C shows that downregulation of excision repair, double-strand break repair, and crosslink Egr1 abolished the enhanced p73 induction and apoptosis repair. We find that apoptosis and clonogenic cell death after oxaliplatin treatment seen in p53 / Cry1/2 / cells by genotoxicants in p53-null background is enhanced by relative to the p53 / cells. In contrast, and rather strikingly, Cry mutation exclusively for UV mimetics–induced DNA Egr1 downregulation has no significant effect on p73 induction damage and no other. Furthermore, we present evidence and apoptosis by doxorubicin in either background. As pre- indicating that the enhanced apoptosis and clonogenic dicted by the model, downregulation of E2F1 has the opposite death by UV mimetic agents in Cry-mutant background is effect (Fig. 4D and E): Reducing E2F1 does not affect either due to the upregulation of the clock-controlled transcrip- the basal level or the amplified apoptosis induced by oxalipla- tion factor Egr1 in Cry-mutant cells, which lead to high level tin (Fig. 4D) but reduces the doxorubicin-induced p73 induc- of induced expression of p73 tumor suppressor/apoptosis tion and apoptosis in both p53 / and p53 / Cry1/2 / promoter. background (Fig. 4E). To recapitulate, double-strand breaks In cells with functional p53, apoptosis by genotoxic agents upregulate p73 through E2F1, and as E2F1 is not a clock- is mainly controlled by this tumor suppressor (20). Only in controlled gene, clock disruption through Cry mutation does p53-mutant cells, p73 becomes the arbitrator of cell fate by not affect apoptosis induced by doxorubicin. In contrast, intrinsic apoptosis (21). In contrast to p53, DNA damage– oxaliplatin upregulates p73 through Egr1, which has a clock- induced upregulation of p73 is mainly by transcriptional controlled promoter and therefore the overproduced Egr1 induction, although posttranscriptional modifications also in Cry-mutant cells strongly enhances oxaliplatin-induced make some contribution (4–8). The transcriptional regulation apoptosis in p53-mutant cells. of p73 has not been investigated in great detail. Current knowledge may be summarized as follows: The p73 promoter (2,000 bp preceding transcriptional start site) contains, in the Discussion 50 to 30 direction, 5 Egr1-, 1 C-EBPa, and 3 E2F1-binding sites We recently reported that cryptochrome mutation ren- (Fig. 5). Egr1 and E2F1 are transcriptional activators, whereas dered the p53-null and the Ras-transformed p53-null cells, C-EBPa functions as a repressor. In most cell types, in the but not cells with wild-type p53, more susceptible to killing absence of DNA damage, the p73 expression is rather low (8). by agents that activate either the intrinsic or the extrinsic However, all 3 transcription factors regulating p73 expression apoptosis pathways (3, 19). In those studies, UV or oxali- are subject to regulatory mechanisms themselves: Egr1 is platin were used to initiate intrinsic apoptosis (3) and TNFa controlled by the circadian clock (3, 9), C-EBPa is controlled to initiate extrinsic apoptosis (19). As multiple agents that by posttranslational modification and nucleocytoplasmic damage DNA can initiate intrinsic apoptosis and similarly shuttling, and E2F1 is controlled by the cell cycle and by DNA multiple extracellular factors that influence cellular homeo- damage–induced phosphorylation and acetylation, which sta- stasis can initiate extrinsic apoptosis (14) it is of interest to bilize the protein and enhance its activity (15–18, 22). Upon determine whether intrinsic and extrinsic apoptosis or other DNA damage, C-EBPa is phosphorylated, released from the death forms induced by a variety of agents are similarly p73 promoter, and excluded from the nucleus; E2F1 is acet- potentiated by Cry mutation. Here, we have investigated the ylated and stabilized, leading to 10- to 30-fold induction of p73

UV IR

Circadian Egr1 C-EBPα E2F1 clock p73 5′ 3′ –2243 –1728 –1380 –1372 –155 –132 +1 Egr1 C-EBPα E2F1

Figure 5. Model for clock effects on DNA damage–induced upregulation of p73 tumor suppressor. The 2 kb of the upstream sequence of the p73 promoter is shown in the direction 50 to 30, with the transcription start site indicated with a bent arrow. Functional transcription factor–binding sites are indicated by open boxes, and the transcription factors are shown as circles. Transcription factor–binding sites include 5 early growth response-1 (Egr1) sites, 1 CCAAT/ enhancer-binding protein (C-EBPa) site, and 3 E2F1 sites. Egr1 and E2F1 are transactivators, and C-EBPa functions as a repressor. Both UV and ionizing radiation (IR) cause dissociation of C-EBPa from the promoter. UV-induced upregulation of p73 is mediated by the clock controlled transcription factor Egr1. In contrast, IR-induced p73 upregulation is predominantly controlled by E2F1, which is not affected by the circadian clock.

790 Cancer Res; 73(2) January 15, 2013 Cancer Research

Cryptochrome Mutation and DNA Damage–Induced Apoptosis

transcription from the promoter that is no longer repressed Disclosure of Potential Conflicts of Interest by C-EBPa. Of the 2 positive transcription factors, E2F1 seems No potential conflicts of interest were disclosed. to have the predominant effect. However, the phosphorylation and acetylation of E2F1 are induced by ionizing radiation and Authors' Contributions radiomimetic agents such as doxorubicin but not by UV or UV Conception and design: J.H. Lee, N. Ozturk, A. Sancar mimetic agents such as platinum-based drugs. As a conse- Development of methodology: J.H. Lee, S. Gaddameedhi, N. Ozturk, A. Sancar Acquisition of data (provided animals, acquired and managed patients, quence, it might be expected that in our experimental system, provided facilities, etc.): N. Ozturk, A. Sancar doxorubicin-induced p73 upregulation, which is predominant- Analysis and interpretation of data (e.g., statistical analysis, biostatistics, computational analysis): J.H. Lee, S. Gaddameedhi, N. Ozturk, A. Sancar ly controlled by E2F1 would not be affected by the presence or Writing, review, and/or revision of the manuscript: J.H. Lee, S. Gadda- absence of a functional circadian clock because E2F1 is not meedhi, N. Ozturk, R. Ye, A. Sancar controlled by the clock. In contrast, upregulation of p73 by UV Administrative, technical, or material support (i.e., reporting or orga- nizing data, constructing databases): N. Ozturk, A. Sancar and UV mimetic agents seems to be mediated primarily by Egr1 Study supervision: N. Ozturk, A. Sancar because UV does not affect posttranslational modification of E2F1 and its activity (16–18). Furthermore, as Egr1 is under robust clock control (3, 9), in Cry mutants in which the Grant Support – This work was supported by NIH grants (GM31082 and GM32833) to A. inhibitory arm of the transcription translation feedback loop Sancar. is nonfunctional, Egr1 is highly elevated leading to significantly The costs of publication of this article were defrayed in part by the enhanced DNA damage–inducible p73 transcription even in payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate the absence of contribution from E2F1. Because doxorubicin this fact. plus oxaliplatin combination is included in several chemother- fi apy regimens, these ndings should be taken into account for Received May 21, 2012; revised October 5, 2012; accepted October 6, 2012; optimal efficacy with minimal side effects. published OnlineFirst November 13, 2012.

References 1. Reppert SM, Weaver DR. Coordination of circadian timing in mam- 11. Sancar A, Lindsey-Boltz LA, Unsal-Kacmaz K, Linn S. Molecular mals. Nature 2002;418:935–41. mechanisms of mammalian DNA repair and the DNA damage check- 2. Ozturk N, Lee JH, Gaddameedhi S, Sancar A. Loss of cryptochrome points. Annu Rev Biochem 2004;73:39–85. reduces cancer risk in p53 mutant mice. Proc Natl Acad Sci U S A 12. Antoch MP, Kondratov RV. Circadian proteins and genotoxic stress 2009;106:2841–6. response. Circ Res 2010;106:68–78. 3. Lee JH, Sancar A. Circadian clock disruption improves the efficacy of 13. Kelland L. The resurgence of platinum-based cancer chemotherapy. chemotherapy through p73-mediated apoptosis. Proc Natl Acad Sci Nat Rev Cancer 2007;7:573–84. U S A 2011;108:10668–72. 14. Hotchkiss RS, Strasser A, McDunn JE, Swanson PE. Cell death. N Engl 4. Marabese M, Vikhanskaya F, Rainelli C, Sakai T, Broggini M. DNA J Med 2009;361:1570–83. damage induces transcriptional activation of p73 by removing C- 15. Lin WC, Lin FT, Nevins JR. Selective induction of E2F1 in response to EBPalpha repression on E2F1. Nucleic Acids Res 2003;31:6624–32. DNA damage, mediated by ATM-dependent phosphorylation. Genes 5. Urist M, Tanaka T, Poyurovsky MV, Prives C. p73 induction after DNA Dev 2001;15:1833–44. damage is regulated by checkpoint kinases Chk1 and Chk2. Genes 16. Pediconi N, Guerrieri F, Vossio S, Bruno T, Belloni L, Schinzari V, et al. Dev 2004;18:3041–54. hSirT1-dependent regulation of the PCAF-E2F1-p73 apoptotic path- 6. Seelan RS, Irwin M, van der Stoop P, Qian C, Kaelin WG Jr, Liu W. The way in response to DNA damage. Mol Cell Biol 2009;29:1989–98. human p73 promoter: characterization and identification of functional 17. Carnevale J, Palander O, Seifried LA, Dick FA. DNA damage signals E2F binding sites. Neoplasia 2002;4:195–203. through differentially modified E2F1 molecules to induce apoptosis. 7. Pignatelli M, Luna-Medina R, Perez-Rendon A, Santos A, Perez- Mol Cell Biol 2012;32:900–12. Castillo A. The transcription factor early growth response factor-1 18. Biswas AK, Johnson DG. Transcriptional and nontranscriptional func- (EGR-1) promotes apoptosis of neuroblastoma cells. Biochem tions of E2F1 in response to DNA damage. Cancer Res 2012;72:13–7. J 2003;373:739–46. 19. Lee JH, Sancar A. Regulation of apoptosis by the circadian clock 8. Yu J, Baron V, Mercola D, Mustelin T, Adamson ED. A network of p73, through NF-kappaB signaling. Proc Natl Acad Sci U S A 2011;108: p53 and Egr1 is required for efficient apoptosis in tumor cells. Cell 12036–41. Death Differ 2007;14:436–46. 20. Lowe SW, Ruley HE, Jacks T, Housman DE. p53-dependent apo- 9. Bai L, Zimmer S, Rickes O, Rohleder N, Holthues H, Engel L, et al. Daily ptosis modulates the cytotoxicity of anticancer agents. Cell 1993; oscillation of gene expression in the retina is phase-advanced with 74:957–67. respect to the pineal gland. Brain Res 2008;1203:89–96. 21. Irwin MS, Kondo K, Marin MC, Cheng LS, Hahn WC, Kaelin WG Jr. 10. Hughes ME, DiTacchio L, Hayes KR, Vollmers C, Pulivarthy S, Baggs Chemosensitivity linked to p73 function. Cancer Cell 2003;3:403–10. JE, et al. Harmonics of circadian gene transcription in mammals. PLoS 22. Nevins JR. E2F: a link between the Rb tumor suppressor protein and Genet 2009;5:e1000442. viral oncoproteins. Science 1992;258:424–9.

www.aacrjournals.org Cancer Res; 73(2) January 15, 2013 791

DNA Damage−Specific Control of Cell Death by Cryptochrome in p53-Mutant Ras−Transformed Cells

Jin Hyup Lee, Shobhan Gaddameedhi, Nuri Ozturk, et al.

Cancer Res 2013;73:785-791. Published OnlineFirst November 13, 2012.

Updated version Access the most recent version of this article at: doi:10.1158/0008-5472.CAN-12-1994

Supplementary Access the most recent supplemental material at: Material http://cancerres.aacrjournals.org/content/suppl/2012/11/12/0008-5472.CAN-12-1994.DC1

Cited articles This article cites 22 articles, 10 of which you can access for free at: http://cancerres.aacrjournals.org/content/73/2/785.full#ref-list-1

Citing articles This article has been cited by 3 HighWire-hosted articles. Access the articles at: http://cancerres.aacrjournals.org/content/73/2/785.full#related-urls

E-mail alerts Sign up to receive free email-alerts related to this article or journal.

Reprints and To order reprints of this article or to subscribe to the journal, contact the AACR Publications Department at Subscriptions [email protected].

Permissions To request permission to re-use all or part of this article, use this link http://cancerres.aacrjournals.org/content/73/2/785. Click on "Request Permissions" which will take you to the Copyright Clearance Center's (CCC) Rightslink site.