Key role of ATF3 in p53-dependent DR5 induction upon DNA damage of human colon cancer cells

Abstract

Stress response gene ATF3 is one of the p53 target genes and has a tumor suppressor role in cancer. However, the biological role of p53–ATF3 pathway is not well understood. Death receptor 5 (DR5) is a death domain-containing transmembrane receptor that triggers cell death upon binding to its ligand TRAIL (tumor necrosis factor-related apoptosis-inducing ligand), and a combination of TRAIL and agents that increase the expression of DR5 is expected as a novel anticancer therapy. In this report, we demonstrate that ATF3 is required for efficient DR5 induction upon DNA damage by camptothecin (CPT) in colorectal cancer cells. In the absence of ATF3, induction of DR5 messenger RNA and protein is remarkably abrogated, and this is associated with reduced cell death by TRAIL and CPT. By contrast, exogenous expression of ATF3 causes more rapid and elevated expression of DR5, resulting in enhanced sensitivity to apoptotic cell death by TRAIL/CPT. Reporter assay and DNA affinity precipitation assay demonstrate that at least three ATF/CRE motifs at the proximal promoter of the human DR5 gene are involved in the activation of DNA damage-induced DR5 gene transcription. Furthermore, ATF3 is shown to interact with p53 to form a complex on the DR5 gene by Re-chromatin immunoprecipitation assay. Taken together, our results provide a novel insight into the role of ATF3 as an essential co-transcription factor for p53 upon DNA damage, and this may represent a useful biomarker for TRAIL-based anticancer therapy.

Introduction

Apoptosis is a natural physiological process of programmed cell death that helps to eliminate irreversibly damaged cells. Deregulation or resistance of transformed cells to apoptosis contributes to the development of cancers (Hanahan and Weinberg, 2000), and current cancer therapeutics partly aim to enhance programmed cell death (Sellers and Fisher, 1999; Nicholson, 2000). In colorectal cancers, an increased cell death is directly correlated with tumor regression after radiation or chemotherapy (Scott et al., 1998; Farczadi et al., 1999). Thus, understanding the molecular mechanisms that result in cancer cell death may lead to the development of novel cancer therapeutic approaches against colorectal cancer.

Apo2L/TRAIL (tumor necrosis factor-related apoptosis-inducing ligand) is a member of the tumor necrosis factor cytokine family and expressed as a type II transmembrane glycoprotein (Wiley et al., 1995; Pitti et al., 1996). On binding to functional receptors death receptor (DR)4 or DR5, Apo2L/TRAIL causes trimerization of the receptors required for the recruitment of an adaptor protein FADD and the formation of DISC through recruiting the initiator caspase-8 or -10 (Kischkel et al., 2000; Sprick et al., 2000; Wang et al., 2001), resulting in apoptotic death of cancer cells without significant toxicity toward normal cells. This extrinsic pathway is p53-independent, and in some cells, death receptor engagement suffices for commitment to apoptotic cell death. DRs can also activate the intrinsic pathway by caspase-8-mediated cleavage of the pro-apoptotic BCL2 member BID, thus causing its interaction with BAX and BAK, which results in the release of mitochondrial cytochrome c to activate caspase-9 and -3 (Eskes et al., 2000; Wei et al., 2000; LeBlanc et al., 2002). In other cell types, however, commitment to apoptosis requires amplification of extrinsic death receptor signal by the cell-intrinsic pathway, which usually involves p53. DNA damage induced by radio- and chemotherapeutic agents activates p53, which in turn activates different pro-and antiapoptotic Bcl-2 family proteins. Relative ratio of these BcL-2 family proteins determines ultimate sensitivity of cancer cells to apoptosis (Green and Evan, 2002). Thus, a combination therapy of Apo2L/TRAIL or its agonizts with several reagents that enhance the intrinsic pathway is considered to be the potential therapeutic interventions (Ashkenazi and Herbst, 2008).

Camptothecin (CPT) is a potent DNA topoisomerase I inhibitor that stall DNA replication by causing single- and double-strand DNA breaks, resulting in cell cycle arrest at G2/M phase. In fact, for metastatic colon cancer and ovarian cancer, CPT is a key component of the first or second line treatment (Fuchs et al., 2006; Legarza and Yang, 2006). When used in combination with Apo2L/TRAIL, CPT reversed cell cycle arrest and greatly sensitized colon cancer cells to Apo2L/TRAIL-mediated apoptosis or eliminated metastasis both in vitro and in vivo (Gliniak and Le, 1999; LeBlanc et al., 2002; Naka et al., 2002; Xiang et al., 2002; Wang and El-Deiry, 2003). The expression level of DR5 or DR4 correlates with increased responsiveness to Apo2L/TRAIL-mediated apoptosis, and selective induction of DR5 or DR4 gene transcription by p53-dependent pathway has been reported in a number of cancer cell lines treated with CPT (Wu et al., 1997; Sheikh et al., 1998; LeBlanc et al., 2002; Xiang et al., 2002). Furthermore, Takimoto and El-Deiry reported that the human DR5 gene is transactivated by p53 through an intronic p53-binding DNA motif (Takimoto and El-Deiry, 2000). However, the role of other transcription factor(s) that co-regulates with p53 to modulate DR5 expression remains elusive.

The stress-inducible transcription factor ATF3 is a member of the ATF/CREB family of basic-leucine zipper (bZip) type transcription factors. Its messenger RNA (mRNA) level is low or undetectable, but is greatly induced upon exposure of cells to a variety of stress signals, including genotoxic agents such as CPT, doxorubicin and etoposide, as well as cytokines and growth factors (Hai et al., 1999 and references herein). Remarkably, ATF3 has a dichotomous role in determining cell fate. For example, it has a positive role in cell proliferation (Hsu et al., 1991; Tamura et al., 2005) and oncogenesis (Yin et al., 2008; Wu et al., 2010). By contrast, upon DNA damage, ATF3 is induced downstream of p53 and its expression is correlated with apoptotic cell death (Amundson et al., 1999; Fan et al., 2002; Zhang et al., 2002). Also, ATF3 has been shown to be a direct target of p53 (Amundson et al., 1999; Fan et al., 2002; Zhang et al., 2002; Wei et al., 2006; Riley et al., 2008). Furthermore, of intrigue, Yan et al. demonstrated that ATF3 protein directly interacts with p53 under genotoxic stress and stabilizes p53 by inhibiting its ubiquitylation (Yan et al., 2005). More recently, physical interaction of ATF3 with p53 is reported by a global analysis of interaction of transcription factors (Ravasi et al., 2010). Taken together, these may imply a possible role of ATF3 in apoptosis, cell cycle arrest and DNA repair by regulating p53. However, at this moment, our knowledge on the cooperative role of ATF3 with p53 at the transcription regulation is limited.

Here, we report that the stress response gene ATF3 acts as a transcriptional activator of DR5 expression by CPT in human colorectal cancer cells, and is an essential co-transcription factor for p53 to activate the DR5 gene promoter, providing an insight into the biological role of p53–ATF3 pathway. This study also describes evidence for the implication of ATF3 in TRAIL-induced apoptosis of cancer cells, and ATF3 may represent a novel biomarker of TRAIL-based therapeutic approach.

Results

CPT causes p53-dependent induction of ATF3 and DR5 in HCT116 cells

In our search for target genes of ATF3 using genome-wide Chromatin immunoprecipitation (ChIP)-on-chip screening, ATF3 was significantly recruited onto the proximal promoter of the DR5 gene upon DNA damage (GEO: accession number GSE18457, http://www.ncbi.nlm.nih.gov/geo/). To understand the role of ATF3 in DR5 induction, we examined the expression of ATF3 and DR5 in CPT-treated human colon cancer HCT116 cells. As shown in Figure 1a, both ATF3 and DR5 were upregulated following the p53 stabilization in cells with wild-type (wt) p53 gene, but this induction was significantly suppressed in HCT116 p53 null cells. Figure 1b shows that ATF3 mRNA was more rapidly induced with a peak at 6 h after treatment and then declined. By contrast, DR5 mRNA continued to increase until 24 h. These results indicate that CPT causes p53-dependent upregulation of ATF3 and DR5, suggesting that ATF3 might have a role in DR5 induction. DR4 mRNA was also upregulated in a p53-dependent manner (Supplementary Figure 1a).

Figure 1
figure1

CPT-induced expression of DR5 and ATF3 depends on p53. (a) HCT116 p53 wt and p53 null cells were treated with 0.2 μM CPT for the indicated time, and their whole-cell extracts were analyzed for DR5 and ATF3 expressions by western blot. Density of each band was measured and its relative expression to β-actin is shown. (b) ATF3 and DR5 mRNA levels of cells as treated in (a) were determined by qRT–PCR. Relative expression of the transcripts was normalized to GAPDH.

ATF3 is required for DR5 induction upon DNA damage in HCT116 cells and in other cancer cells

Next, we addressed if ATF3 has a role in DR5 induction. To this end, we employed loss of function of gene approach using small interfering RNAs specific for ATF3. As shown in Figure 2, knockdown of ATF3 significantly suppressed the induction of DR5 protein and mRNA (Figure 2a) in HCT116 p53 wt cells, compared with control small interfering RNA. We further examined the effect of ATF3 knockdown in HCT116 p53 null cells as both ATF3 and DR5 were still induced by CPT in these cells (Figure 1a). Figure 2b shows that the induction of DR5 protein and mRNA was suppressed by knockdown of ATF3. These data demonstrate that the DR5 gene is induced by CPT in both p53-dependent and -independent manner, and ATF3 has a role in both pathways. Knockdown of ATF3 also abrogated the CPT-induced upregulation of DR4 mRNA (Supplementary Figures 1b and c). We next examined if ATF3 is required for DR5 induction by doxorubicin and etoposide, as these agents are known to induce ATF3 and DR5. Figure 2c shows that knockdown of ATF3 significantly inhibited the induction of DR5 mRNA by doxorubicin and etoposide in HCT116 cells. Further, the DR5 induction by CPT was significantly abrogated by knockdown of ATF3 in other human cancer LoVo cells and MCF7 cells (Figure 2d). Thus, it is supported that ATF3 is required to induce the DR5 gene transcription upon DNA damage in various cancer cells. As shown in Supplementary Figures 2a and b, the induction of DR4 upon DNA damage was also suppressed by ATF3 knockdown.

Figure 2
figure2

DR5 induction is regulated by ATF3 upon DNA damage in several cell lines. (a, b) HCT116 p53 wt and HCT116 p53 null cells were transfected with siControl or siATF3 oligos. After treatment with 0.2 μM CPT, expressions of DR5 and ATF3 proteins or mRNAs were analyzed by western blot or qRT–PCR, respectively. Relative expressions are shown as in Figures 1a and b. (c) HCT116 p53 wt cells transfected with siControl or siATF3 oligos were untreated, or treated with 1.0 μM doxorubicin (DXR) or 30 μM etoposide (VP-16) for 12 h, and then ATF3 and DR5 mRNA levels were determined by qRT–PCR. (d) LoVo cells and MCF7 cells transfected with siControl or siATF3 oligos were treated with 0.2 μM CPT for 24 h. ATF3 and DR5 mRNA levels were determined as in (c). Relative expressions of these transcripts were normalized to GAPDH. *P<0.05; **P<0.01.

Loss of p53 and atf3 strongly inhibits CPT-induced DR5 expression in mouse embryonic fibroblasts (MEFs)

In order to confirm the role of ATF3 for the induction of DR5 more clearly, we generated atf3 knockout mice using Cre-loxP recombination system (Figures 3a–c). MEFs prepared from atf3 null or p53 null mice were treated with CPT in parallel, and DR5 induction was examined. Figure 3d shows that, in p53 null MEFs, upregulation of atf3 mRNA (left panel) and dr5 mRNA (right panel) are remarkably suppressed, indicating both genes were activated downstream of p53 in response to CPT. In atf3 null MEFs (Figure 3e), the induction of dr5 mRNA was significantly suppressed (left panel) as well. When the expression of ATF3 was re-introduced into the atf3 null MEFs, the induction of dr5 mRNA was recovered to almost wt level (right panel). Similarly, the induction of DR5 protein by CPT was less in p53 null and atf3 null MEFs (Figure 3f left panel). It should be noted that CPT-induced DR5 protein level was clearly restored in ATF3 re-introduced MEFs (Figure 3f right panel). These data strongly support that ATF3 is a positive regulator of DR5 induction upon DNA damage.

Figure 3
figure3

Generation of atf3 knockout mice and loss of p53 or atf3 gene abrogates DR5 induction in MEFs. (a) Schematic diagram of the generation of conditional and constitutive atf3 knockout mice. Positions of selected restriction enzymes and of coding exon 2 (E2) as well as of loxP recombination sites (triangles) are indicated (neo: neomycin resistance cassette; DT-A: diphtheria toxin). The targeting strategy and primer sequences are described in Materials and methods. (b) Southern hybridization of ES cells treated with adenovirus expressing Cre-recombinase. Cellular DNA samples were digested with SpeI and hybridized with the internal probe as indicated in (a). (c) Wt and atf3 deleted genome DNA eluted from MEFs were amplified by PCR with two primer sets (#1 and #3) indicated in (a) (left panel). MEFs were treated with 2.0 μM CPT, and ATF3 expression was assessed by western blotting (right panel). (d, e) Wt and p53 null MEFs (d) or atf3 null MEFs (e) were treated with 2.0 μM CPT for the indicated time, and assayed for atf3 and dr5 mRNAs by qRT–PCR. In (e), the induction of dr5 mRNA was also assayed after the human ATF3 was re-introduced into atf3 null MEFs as in Materials and methods. Relative expression of transcripts was normalized to GAPDH. (f) Wt and p53 null or atf3 null MEFs were treated with 2.0 μM CPT for 12 h, then, DR5, ATF3 and p53 proteins were analyzed by western blotting.

ATF3 is required for cell death induced by a combination of TRAIL and CPT

TRAIL induces apoptotic cell death upon binding to its receptor DR5 in HCT116 cells, and its activity is augmented by pre-treatment of cells with CPT (Gliniak and Le, 1999; LeBlanc et al., 2002; Naka et al., 2002; Xiang et al., 2002; Wang and El-Deiry, 2003). Thus, we next examined the role of ATF3 in cell death by the combined treatment with TRAIL and CPT. Figure 4 shows that both reagents, when present in combination, markedly induced death of HCT116 cells. By contrast, knockdown of ATF3 significantly inhibited the cell death measured by cell death assay or fluorescence-activated cell sorting analysis (Figures 4a–c). This suppression was accompanied by biochemical evidence that siATF3 downregulated the cleavage of PARP and caspase-3, markers of apoptotic cell death (Figure 4d). We next tested the effect of ATF3 on DR5 expression and TRAIL-induced cell death by overexpressing ATF3. As shown in Figure 4e, exogenous ATF3 expression caused modest upregulation of DR5 in the absence of CPT, but treatment with TRAIL and CPT caused much higher and more rapid induction of DR5 in the ATF3-overexpressing cells than in the control cells. Consistently, more cell death was observed in ATF3-overexpressing cells by TRAIL/CPT. Collectively, it is indicated that ATF3 is required for an increased sensitivity of cells to the combined treatment of TRAIL and CPT.

Figure 4
figure4

ATF3 sensitizes HCT116 cells to TRAIL-induced apoptosis. (a) HCT116 p53 wt cells were transfected with siControl or siATF3 oligos. After 24 h, cells were treated with 0.2 μM CPT with or without 2.5 ng/ml TRAIL for another 24 h. Then, trypan blue exclusion assay was carried out as detailed in Materials and methods, and relative values of dead cell numbers are shown with s.e. from three independent experiments. *P<0.05. (b) Nuclei of HCT116 cells treated as in (a) were visualized using 6-diamidino-2-phenylindole staining under a fluorescence microscope. Arrowheads show fragmented nuclei. (c) HCT116 cells stably expressing siGFP or siATF3-363 were treated with 0.2 μM CPT with or without 2.5 ng/ml TRAIL for 24 h, then fluorescence-activated cell sorting analysis was carried out as in Materials and methods. Relative cell number at sub-G1 fraction was quantitated as sub-G1 population (%). (d) Whole-cell extracts of HCT116 cells treated as in (a) were analyzed by western blotting using anti-PARP, anticaspase3, anticleaved caspase3, anti-ATF3 or anti-DR5 antibodies. (e) HCT116 cells stably expressing ATF3 or GFP were treated with 0.2 μM CPT for the indicated time, and whole-cell extracts were analyzed by western blotting using anti-DR5, anti-ATF3 or anti-p53 antibodies (upper panel). In the middle panel, cells were treated with 0.2 μM CPT with or without 2.5 ng/ml TRAIL for 24 h, and trypan blue exclusion assay was carried out as in (a). Data represent means with s.e. of three independent experiments.*P<0.05. Cells treated as in the middle panel were subjected to fluorescence-activated cell sorting analysis (lower panel) as in (c).

ATF3 is recruited onto the DR5 gene promoter and induces the DR5 gene-luciferase activity in response to CPT

Data above strongly support that ATF3 activates the DR5 gene transcription upon CPT treatment, and our genome-wide ChIP-on-chip analysis demonstrated that ATF3 was recruited onto the DR5 gene promoter upon DNA damage (Supplementary Figure 3). To delineate the cis-element(s) responsible for the DR5 induction by ATF3, we first examined the in vivo recruitment of ATF3 onto the possible ATF/CRE motifs in the human DR5 gene as illustrated in Figure 5a. Among the six motifs tested, ATF3 was indeed recruited onto the ATF-BS2, 3 and 4 sites in response to CPT (Figure 5b). p53 was recruited to the p53-BS as reported (Takimoto and El-Deiry, 2000). Next, we assayed the promoter activity of the human DR5 gene using various luciferase reporter constructs. Figure 5c showed that pGL2-Full, pDR5-448, -418 and -198, but not further deletion to -168, were clearly activated by CPT, demonstrating that CPT response is mediated by sequence from −418 to −168 containing ATF-BS3 and 4. Thus, a set of mutations of BS3 or BS4 of the pDR5-418 reporter was constructed. As shown in Figure 5d, BS-3 or BS4 mutant alone (3m4w and 3w4m) caused modest but significant reduction, but the mutant of both sites (3m4m) almost abrogated the induction, indicating these two sites are fully required for CPT response. In Figure 5e, the induction of pDR5-418 and pGL2-Full was reduced in ATF3 knocked-down cells. We further examined the effect of ATF3 and p53 expression on the reporter activity using p53-deficient HCT116 cells. As shown in Figure 5f, ATF3 as well as p53, but not mutant p53, induced the reporter activity of pDR5-418. When both p53 and ATF3 were expressed, the synergistic induction of reporter activity was observed, especially in lower expression of p53. This might suggest a predominant role of p53 in DR5 induction. Collectively, these data support that ATF3 is an activator of the DR5 gene expression through its recruitment to ATF/CRE motif, at least, BS3 and BS4 sites on the promoter of the DR5 gene and ATF3 cooperates with p53 in the CPT-induced DR5 gene expression.

Figure 5
figure5

ATF3 is recruited onto the DR5 promoter and activates human DR5 reporter gene. (a) Schematic representation of the human DR5 gene promoter. Six possible ATF/CREB binding sites (ATF-BS1 through -6) and a p53 binding site (p53-BS) are shown along with matrix score (Software Genomatix, Munich, Germany). (b) HCT116 p53 wt cells were treated with 1.0 μM CPT for 12 h, then ChIP assay combined with standard PCR was performed using anti-ATF3 or anti-p53 antibodies, detailed as in Materials and methods. (c) Cells were transfected with 0.5 μg each of pGL2-Full or various deletions as indicated for 16 h. After treatment with 1.0 μM CPT for 12 h, cell extracts were assayed for luciferase activity. Relative luciferase activity represents the fold induction compared with that of non-stimulated cells transfected with pGL2-Full. (d) Cells were transfected with 0.5 μg each of pDR5-Luc418 reporters containing mutations at ATF-BS3 and/or -4 and treated as in (c). Relative luciferase activity represents the fold induction compared with untreated pDR5-Luc418 (3w4w). (e) Cells whose ATF3 expression was retrovirally knocked down were transfected with pGL2-Full or pDR5-Luc418, treated with 1.0 μM CPT for 12 h, and assayed for luciferase activity. Relative luciferase activity represents the fold induction compared with untreated cells with PGL2-Full. (f) HCT116 cells deficient for p53 allele were co-transfected with 0.5 μg pDR5-Luc418 and indicated effector expression vectors (pCI-ATF3: 0.5 μg, pCMV-p53wt: 0.005 μg, 0.05 μg, 0.5 μg, pCMV-53 mt: 0.5 μg), then assayed for luciferase activity. Relative luciferase activity represents the fold induction compared with that without effector plasmids. All the data are shown as means with s.e. bars of three independent experiments. *P<0.05, significant against no effector expression vector (P<0.05).

ATF3 interacts with ATF/CRE binding motifs of the human DR5 gene promoter in response to CPT and cooperates with p53

To examine direct binding of ATF3 onto the ATF/CRE motifs in the DR5 gene promoter, we performed DNA affinity precipitation (DNAP) assay using biotin-labeled ATF-BS2, 3 and 4 oligonucleotides. As shown in Figure 6a, all of these showed specific binding of ATF3 in CPT-treated HCT116 cells, and more importantly the binding was abrogated with wt, but not mutant probes. It is reported that ATF3 physically interacts with p53 under genotoxic stress (Yan et al., 2005; Ravasi et al., 2010), thus, we measured the interaction of ATF3 with p53 in CPT-treated cells. Figure 6b shows that immunoprecipitation of ATF3 specifically pulled down p53 (left panel) and vice versa (right panel). Next, we explored a possible interaction of ATF3 with p53 on the DR5 gene promoter as their respective binding sites are closely located to each other. In Figure 6c, re-ChIP assays clearly show that ATF-BS2, 3 and 4 were specifically enriched in the chromatin complexes immune-isolated by anti-p53 antibody, followed by anti-ATF3 antibody and vice versa, suggesting that ATF3 and p53 are present in the same chromatin complexes on the DR5 gene promoter. These data, altogether, support that ATF3 binds to ATF/CRE motifs of the DR5 gene promoter and also interacts with p53 in the intron 1, providing evidence for integrated chromatin complex of ATF3 with p53.

Figure 6
figure6

ATF3 binds to the ATF/CRE motifs of the human DR5 gene promoter and interacts with p53. (a) Nuclear extracts of HCT116 p53 wt cells were prepared after treatment with 0.2 μM CPT for 24 h, and subjected to DNAP assay using biotinylated ATF-BS2, BS3 or BS4 probes as detailed in Materials and methods (upper panel). In the lower panel, ten times molar excess of wt or mutant-type(mt) oligonucleotide was used as competitor. (b) Whole lysates of cells treated as in (a) were incubated with 1 μg each of anti-ATF3 or anti-p53 antibody overnight, and immunoprecipitates were captured and subjected to western blotting. Lysates were also immunoprecipitated with 1 μg normal IgG as a control. Arrows indicate specific bands. Arrowheads denote IgG. (c) Cells treated as in (a) were subjected to Re-ChIP assay. Briefly, the first ChIP was performed by 1 μg each of anti-ATF3 or anti-p53 antibody, and the eluted samples were sequentially immunoprecipitated with the second antibody. Lysates were also immunoprecipitated with 1 μg normal IgG as a control. Precipitated chromatin was analyzed by PCR using primer sequences flanking the ATF3 or p53 binding site as in Materials and methods. Primer set for GAPDH was also used as a negative control.

Discussion

We and others have previously shown that the stress response gene ATF3 is one of the p53 target genes upon genotoxic stress (Amundson et al., 1999; Fan et al., 2002; Zhang et al., 2002; Wei et al., 2006; Riley et al., 2008); however, its biological significance still remained elusive. In this study, we clearly demonstrate that the DR5 gene is a direct target of ATF3 upon DNA damage, and ATF3 has a key role in apoptosis induced by CPT/TRAIL co-treatment of human colorectal cancer cells.

Upon CPT treatment of human colorectal cancer HCT116 cells, ATF3 was recruited onto at least three ATF/CRE motifs on the DR5 gene promoter, BS2, 3 and 4, which are localized in the proximity to the major transcriptional start sites determined by Yoshida et al. (2001). These ATF3 bindings to the proximal promoter region of the DR5 gene were consistent with data by genome-wide ChIP-on-chip screening of cells treated by MMS (GEO: accession number GSE18457). The sequences of BS2, 3 and 4 are not perfectly matched to the one of ATF/CRE consensus motif, TGACGTCA, but ATF3 specifically bound to all of the motifs in DNAP assay (Figure 6). BS3 and 4 have positive effect in reporter assay, whereas BS2 did not show apparent effect (Figure 5). Thus, it is likely that these sites function like three tandem sites for ATF3 binding, and the deletion of the most 5′ upstream BS2 site had only modest effect on the reporter activity.

In particular, our data support that the p53-ATF3 axis includes multiple interfaces for the induction of the DR5 gene expression. First, as shown previously, p53 stabilized upon DNA damage brings about the efficient transcription of ATF3 gene, as one of the p53 target genes (Amundson et al., 1999; Fan et al., 2002; Zhang et al., 2002). Second, as shown in the Figure 6 of the present study, the IP and re-ChIP data display that both proteins are recruited to the same chromatin complex on the DR5 gene promoter. We thus propose that the protein–protein interaction of p53 and ATF3 may cause two regions, the ATF/CRE site on proximal promoter and the p53 binding site in the intron 1, to come in close proximity, resulting in the facilitated DR5 gene transcription (Figure 7). In this regard, we predict that the intervening region between ATF/CRE and p53 sites is likely to form a gene-loop structure. As the third interface, Yan et al. (2005) reported that ATF3 prevents p53 from MDM2-mediated degradation by blocking its ubiquitylation, thus, knockdown of ATF3 causes inefficient p53 induction and impaired apoptosis. In this study, however, we could not detect significant reduction of p53 protein level upon CPT treatment in ATF3 knocked-down cells (Figure 1c). Collectively, it is highly likely that ATF3 mediates the signal of activated p53 as multi-faceted device. In this report, for the first time, we have provided the evidence that ATF3 is an essential transcription cofactor for p53 in inducing the DR5 gene transcription upon DNA damage. Even so, our present results do not exclude roles for other known factors in the DR5 gene activation. Several transcription factors such as c-Myc, CHOP or SP1 are reported to be involved in DR5 gene expression (Yoshida et al., 2001; Yamaguchi and Wang, 2004; Sussman et al., 2007). Indeed, we quantified CHOP mRNA and found approximately twofold increase in CPT-treated HCT116 cells independently of p53 gene status (data not shown). As CHOP and SP1 binding sites on the human DR5 gene promoter are located close to those for ATF/CRE, these factors probably have a role in DR5 expression in this study. By contrast, c-Myc mRNA or protein did not change significantly (data not shown). However, using genomatix software, we found a candidate E-box motif with score of 0.93 at rather far upstream region of human DR5 gene promoter. The possible implication of these factors in DR5 gene activation must await further study.

Figure 7
figure7

A schematic model of ATF3 as a co-transcription factor for p53 in DNA damage-induced expression of the DR5 gene. DNA damage activates p53 and its target ATF3 gene through a transcriptional cascade. Both proteins are cooperatively recruited onto the DR5 gene promoter and are involved in the formation of competent transcriptional complex via protein–protein interaction, causing efficient activation of the DR5 gene transcription. This interaction may have a crucial role in enhancing cell death by TRAIL-based combination therapy with genotoxic anticancer agents.

It is worth considering the extraordinary complexity of the ATF3 function in vivo. For example, ATF3 has been reported to have a dichotomous role in cancer, as tumor suppressor or oncogenic gene (Yin et al., 2008). Indeed, ATF3 is correlated with tumor growth and metastasis in various cancers, such as prostate cancer (Pelzer et al., 2006), Hodgkin disease (Janz et al., 2006), mammary cancer (Yin et al., 2008) and more recently skin cancer (Wu et al., 2010). We have previously reported that ATF3 downregulates p53 at the transcription level in primary human umbilical vein endothelial cells (Kawauchi et al., 2002) or cardiomyocytes (Nobori et al., 2002). This ATF3–p53 negative feedback is also reported in skin cancer (Wu et al., 2010). However, the present study is consistent with the role of ATF3 as a tumor suppressor by upregulating DR5 in HCT116 cells under CPT-TRAIL co-treatment to cause cancer cell death. In this regard, it is also reported that ATF3 induces TRAIL in UV-induced apoptosis of skin keratinocytes (Turchi et al., 2008) or diindolylmethane-treated pancreatic cancer cell death (Yoon et al., 2011). In this study, we did not examine if TRAIL was also induced downstream of ATF3 in HCT116 cells, but it is of intrigue to note that ATF3 upregulates not only DR4/5 but also its ligand TRAIL, synergistically enhancing TRAIL-dependent cell death in vivo. Indeed, Hackl et al. (2010) recently reported that HCT116 cells whose ATF3 expression is stably knocked down significantly enhance tumor growth and metastasis of cells in xenografts, supporting that ATF3 functions as a tumor suppressor in colorectal cancer cells.

Although some cancer cells are resistant to TRAIL therapy, combination anticancer therapy by TRAIL-based approach can generally result in an additive tumoricidal effect due to the activation of independent stress pathways. In this report, upon co-treatment by TRAIL/CPT, ATF3 has a pro-apoptotic role in a p53-dependent manner. It is noteworthy that DR5 is upregulated by overexpression of c-Myc or oncogenic Ras mutants, resulting in an enhanced sensitivity of cells to TRAIL-induced death (Nesterov et al., 2004; Wang et al., 2004), as we and other groups reported that ATF3 is expressed downstream of c-myc (Tamura et al., 2005) and oncogenic Ras (Lu et al., 2006; Miyazaki et al., 2009). While it is still unclear how c-Myc or oncogenic Ras regulate TRAIL receptor signaling, ATF3 may be involved in the induction of DR5 by c-Myc or Ras. This remarkable connection between ATF3 and other cancer-related genes must necessitate a clear delineation.

Finally, we demonstrated that ATF3 is a pro-apoptotic transcription factor that cooperates with p53 on the DR5 gene promoter, and has a key role in cell death by TRAIL–CPT co-treatment. ATF3 may be used as a novel biomarker for sensitivity of cells to Apo2/TRAIL-based therapy, and a tool to discover the truly effective agents for the TRAIL-combined anticancer therapy.

Materials and methods

Plasmids, antibodies and reagents

Expression plasmids encoding human ATF3 and human wt or mutant p53 were as described (Cai et al., 2000; Zhang et al., 2002). Retrovirus vector for human ATF3 was constructed using pMX-puromycin vector provided from Dr Kitamura. Antibodies used were as follow: anti-ATF3 (C19), anti-p53 (DO-1 and FL-393) from Santa Cruz Biotechnology (Santa Cruz, CA, USA), anti-β-actin (AC-74) from Sigma-Aldrich (St Louis, MO, USA), anti-DR5 from ProSci (San Diego, CA, USA), anti-PARP from R&D systems (Minneapolis, MN, USA), anticaspase3 from BD Transduction Laboratories (Franklin Lakes, NJ, USA) and anticleaved caspase-3 from Cell Signaling Technology (Beverly, MA, USA). CPT, doxorubicin and etoposide were purchased from Sigma-Aldrich, and recombinant human APO2/TRAIL was from PeproTech (Rocky Hill, NJ, USA). Other chemicals were reagents grade.

Cell culture and medium

HCT116 cells with wild p53 allele, LoVo cells and MCF7 cells were obtained from American Type Culture Collection (Manassas, VA, USA). HCT116 cells with null p53 allele were kindly provided from Dr Vogelstein. Culture media used were McCoy medium for HCT116 cells, Dulbecco's modified Eagle's medium for LoVo and RPMI medium for MCF7.

Generation of mutant mice and MEFs

Knockout mice deficient for atf3 gene were produced by homologous recombination in embryonic stem cells. The targeting vector contained the following elements: (1) a loxP site was inserted into the 5′ intron of exon 2; (2) a neomycin gene cassette surrounded by two loxP sites were inserted into the 3′ intron of exon 2; and (3) a diphtheria toxin gene cassette was placed next to the homologous long-arm sequence. This targeting vector was electroporated into embryonic RW4 stem cells, positive clones were selected by G418, clones harboring homologously recombined atf3 mutant alleles were identified by Southern blotting and mice were generated from these clones by blastocyst injections. The floxed mice were then crossed with EIIa-Cre mice expressing Cre-recombinase in germ cells. Constitutive atf3 knockout mice were genotyped by PCR using the following primers: #1, 5′-IndexTermACTGGGGCAAAGAAACATACC-3′ (forward) and 5′-IndexTermAAAAAGAATCGGGAAGACACT-3′ (reverse); #3, 5′-IndexTermGGTGTGTTTACCTTCTTCATT-3′ (forward) and 5′-IndexTermTCTTGATCTTCCTGTTTCAGT-3′ (reverse). EIIa-Cre mice were purchased from Jackson Labs (Bar Harbar, ME, USA), and p53 knockout mice were obtained as described (Donehower et al., 1992). MEFs were isolated by trypsinization of embryos dissected on the day 13.5 of gestation, and cultured in Dulbecco's modified Eagle's medium. All animal experiments were done with the approval of the Institutional Animal Care Committee of the Tokyo Medical and Dental University, and Osaka Medical Center for Cancer and Cardiovascular Diseases.

Whole-cell extracts and western blot analysis

Whole-cell extracts were prepared and subjected to western blot as described (Cai et al., 2000). Densitometric measurement of bands was performed by scanning immunoblot images using the Image J software (Bethesda, MD, USA).

Quantitative reverse transcription (qRT)–PCR

qRT–PCR was performed as described (Miyazaki et al., 2009). The following primer pairs were employed: human ATF3, 5′-IndexTermCTCCTGGGTCACTGGTGTT-3′ (forward) and 5′-IndexTermTCTGAGCCTTCAGTTCAGCA-3′ (reverse); human DR5, 5′-IndexTermCAGGTGTCAACATGTTGTCC-3′ (forward) and 5′-IndexTermATCGAAGCACTGTCTCAGAG-3′ (reverse); human DR4, 5′-IndexTermACAGGTGTCACTGTACAGTC-3′ (forward) and 5′-IndexTermAGGGCACGATGTTTGCAAAC-3′ (reverse); human glyceraldehyde 3-phosphate dehydrogenase (GAPDH), 5′-IndexTermGAGTCAACGGATTTGGTCGT-3′ (forward) and 5′-IndexTermTTGATTTTGGAGGGATCTCG-3′ (reverse); mouse ATF3, 5′-IndexTermTTACCGTCAACAACAGACCC-3′ (forward) and 5′-IndexTermTCAGCTCAGCATTCACACTC-3′ (reverse); mouse DR5, 5′-IndexTermGAGGCAATGGTTGCTCTGTA-3′ (forward) and 5′-IndexTermCTATGTCCGAACAATACTCG-3′ (reverse); mouse GAPDH, 5′-IndexTermCGTCCCGTAGACAAAATGGT-3′ (forward), 5′-IndexTermTTGATGTTAGTGGGGTCTCG-3′ (reverse). GAPDH was used as an internal control. Data represent means with s.e. bars of three independent experiments.

Knockdown of ATF3

Mixture of small interfering RNA oligos specific for ATF3 (SMARTpool, Dharmacon, Lafayette, CO, USA) were transfected into cells using X-tremeGENE transfection reagent (Roche, Basel, Switzerland). Control RNAi (AM4611) was a silencer control from Ambion (Applied Bioscience, Carlsbad, CA, USA). Retrovirus vector siATF3-363 was constructed by subcloning an oligonucleotide 363, 5′-IndexTermTGGAAAGTGTGTGAATGCTGAACT-3′, into pMX-puroII-U6 (Morita et al., 2000). Cells were first transfected with pcDNA-mcat-encoding ecotropic retrovirus receptor for 48 h, followed by infection with retrovirus for siATF3-363 or siGFP as control, then selected in 2 μg/ml puromycin for several days.

Trypan blue exclusion assay, morphological analysis and fluorescence-activated cell sorting analysis

Trypan blue exclusion assay and fluorescence-activated cell sorting analysis were performed as described (Kawauchi et al., 2002). For morphological analysis, cells were fixed in methanol, incubated with 4′, 6-diamidino-2-phenylindole, and then analyzed using a fluorescent microscope (Olympus, Tokyo, Japan) at 420 nm.

Luciferase assay

Reporter plasmids pDR5Luc-448, -418, -198, -168 and +1 with wt or mutant p53 binding site were prepared by subcloning each DNA fragments into pGL3 vector (Promega, Madison, WI, USA). A set of pDR5Luc-418 reporter plasmids containing mutations of ATF-BS3 or -BS4 were prepared by an overlap extension PCR protocol (Pan and McEver, 1993). pGL2-Full and pDR5PF, and its 5′ deletions were kind gifts from Dr El-Deiry and Dr Sakai, respectively. Luciferase activity was measured using a Dual Luciferase Reporter Assay System from Promega as described (Cai et al., 2000; Tamura et al., 2005). Data represent means with s.e. bars of three independent experiments.

Immunoprecipitation

Nuclear extracts (60 μg of protein) prepared from HCT116 cells were incubated with 1 μg anti-ATF3 or anti-p53 antibodies, respectively, and the immune complexes were washed, eluted and subjected to western blot.

ChIP and re-ChIP

ChIP assays were performed as described (Tamura et al., 2005) using the following primer pairs: ATF3-BS1, 5′-IndexTermAGGCAGCTCCACCTGCAAC-3′ (forward) and 5′-IndexTermGATACACAGTGCCAATTGGTG-3′ (reverse), ATF3-BS2, 5′-IndexTermAGCGACTCTGAACCTCAAGA-3′ (forward) and 5′-IndexTermGTGGTTTGTTTCTGGGTCCTG-3′ (reverse), ATF3-BS3, 5′-IndexTermAAGGTTAGTTCCGGTCCTTC-3′ (forward) and 5′-IndexTermTTCCACCACAGGTTGGTGAC-3′ (reverse), ATF3-BS4, 5′-IndexTermGCAGTTGCACATTGGATCTG-3′ (forward) and 5′-IndexTermTATGTGTCCAGGCTGACTTG-3′ (reverse), ATF3-BS5, 5′-IndexTermAAAGAAAGCCACAACAGCCG-3′ (forward) and 5′-IndexTermCTCTGCTCACTGCAACTTCT-3′ (reverse), ATF3-BS6, 5′-IndexTermCCTCCCAAAATGCTGGGATT-3′ (forward) and 5′-IndexTermAAGAGCCCTCACAGGGAAAT-3′ (reverse), p53-BS, 5′-IndexTermAAGACCCTTGTGCTCGTTGT-3′ (forward) and 5′-IndexTermCGGGAATTTACACCAAGTGG-3′ (reverse), GAPDH, 5′-IndexTermCTTGACTCCCTAGTGTCCTTC-3′ (forward) and 5′-IndexTermAAGGTCTTGAGGCCT-3′ (reverse). In re-ChIP assay, chromatin complexes immunoprecipitated by the first antibody were eluted by 10 mM dithiothreitol. After centrifugation, the supernatant was diluted 20 times with re-ChIP buffer (1% Triton X-100, 2 mM ethylenediaminetetraacetic acid, 150 mM NaCl, 20 mM Tris-HCl, and pH8.0) and subjected to the ChIP procedure by the second antibody.

DNAP assay

DNAP assay was performed as described (Suzuki et al., 1993). Briefly, nuclear extracts (100 μg) from HCT116 cells (Cai et al., 2000) were mixed with biotinylated ATF/CRE probe (100 pmol) in a buffer containing 1 μg poly(dI-dC). Streptavidin agarose beads (Sigma-Aldrich) were added and mixed gently for 1 h. Beads were collected and washed twice, and the bound proteins were analyzed by western blotting. Sequences of ATF/CRE probes were as follows with binding motifs underlined. Wt BS-2 probe, 5′-IndexTermACCCCGGGAGGCGTCAACTCCCCA-3′, mutant BS-2 probe, 5′-IndexTermACCCCGGGAGGAGACAACTCCCCA-3′; wt BS-3 probe, 5′-IndexTermGATTGCGTTGACGAGACTCTTATT-3′, mutant BS-3 probe, 5′-IndexTermGATTGCGTTGTCTAGACTCTTATT-3′; wt BS-4 probe, 5′-IndexTermGCCCCGAATGACGCCTGCCCGGA G-3′, mutant BS-4 probe, 5′-IndexTermGCCCCGAATGTCTCCTGCCCGGA G-3′.

Statistical analysis

Two-tailed Student's t-test was used for statistical analysis of comparative data. Data are presented as mean±s.d., and values of P<0.05 were considered as significant.

Accession codes

Accessions

Gene Expression Omnibus

References

  1. Amundson SA, Bittner M, Chen Y, Trent J, Meltzer P, Fornace Jr AJ . (1999). Fluorescent cDNA microarray hybridization reveals complexity and heterogeneity of cellular genotoxic stress responses. Oncogene 18: 3666–3672.

  2. Ashkenazi A, Herbst RS . (2008). To kill a tumor cell: the potential of proapoptotic receptor agonists. J Clin Invest 118: 1979–1990.

  3. Cai Y, Zhang C, Nawa T, Aso T, Tanaka M, Oshiro S et al. (2000). Homocysteine-responsive ATF3 gene expression in human vascular endothelial cells: activation of c-Jun NH(2)-terminal kinase and promoter response element. Blood 96: 2140–2148.

  4. Donehower LA, Harvey M, Slagle BL, McArthur MJ, Montgomery Jr CA, Butel JS et al. (1992). Mice deficient for p53 are developmentally normal but susceptible to spontaneous tumours. Nature 356: 215–221.

  5. Eskes R, Desagher S, Antonsson B, Martinou JC . (2000). Bid induces the oligomerization and insertion of Bax into the outer mitochondrial membrane. Mol Cell Biol 20: 929–935.

  6. Fan F, Jin S, Amundson SA, Tong T, Fan W, Zhao H et al. (2002). ATF3 induction following DNA damage is regulated by distinct signaling pathways and over-expression of ATF3 protein suppresses cells growth. Oncogene 21: 7488–7496.

  7. Farczadi E, Szanto J, Kaszas I, Benyo I, Bodnar Z, Szlobodnyik J et al. (1999). Changes in apoptotic and mitotic activity in rectal carcinoma after short-term cytostatic therapy as possible predictive factors. Neoplasma 46: 219–223.

  8. Fuchs C, Mitchell EP, Hoff PM . (2006). Irinotecan in the treatment of colorectal cancer. Cancer Treat Rev 32: 491–503.

  9. Gliniak B, Le T . (1999). Tumor necrosis factor-related apoptosis-inducing ligand's antitumor activity in vivo is enhanced by the chemotherapeutic agent CPT-11. Cancer Res 59: 6153–6158.

  10. Green DR, Evan GI . (2002). A matter of life and death. Cancer Cell 1: 19–30.

  11. Hackl C, Lang SA, Moser C, Mori A, Fichtner-Feigl S, Hellerbrand C et al. (2010). Activating transcription factor-3 (ATF3) functions as a tumor suppressor in colon cancer and is up-regulated upon heat-shock protein 90 (HSP90) inhibition. BMC Cancer 10: 668.

  12. Hai T, Wolfgang CD, Marsee DK, Allen AE, Sivaprasad U . (1999). ATF3 and stress responses. Gene Expr 7: 321–335.

  13. Hanahan D, Weinberg RA . (2000). The hallmarks of cancer. Cell 100: 57–70.

  14. Hsu JC, Laz T, Mohn KL, Taub R . (1991). Identification of LRF-1, a leucine-zipper protein that is rapidly and highly induced in regenerating liver. Proc Natl Acad Sci USA 88: 3511–3515.

  15. Janz M, Hummel M, Truss M, Wollert-Wulf B, Mathas S, Johrens K et al. (2006). Classical Hodgkin lymphoma is characterized by high constitutive expression of activating transcription factor 3 (ATF3), which promotes viability of Hodgkin/Reed-Sternberg cells. Blood 107: 2536–2539.

  16. Kawauchi J, Zhang C, Nobori K, Hashimoto Y, Adachi MT, Noda A et al. (2002). Transcriptional repressor activating transcription factor 3 protects human umbilical vein endothelial cells from tumor necrosis factor-alpha-induced apoptosis through down-regulation of p53 transcription. J Biol Chem 277: 39025–39034.

  17. Kischkel FC, Lawrence DA, Chuntharapai A, Schow P, Kim KJ, Ashkenazi A . (2000). Apo2L/TRAIL-dependent recruitment of endogenous FADD and caspase-8 to death receptors 4 and 5. Immunity 12: 611–620.

  18. LeBlanc H, Lawrence D, Varfolomeev E, Totpal K, Morlan J, Schow P et al. (2002). Tumor-cell resistance to death receptor-induced apoptosis through mutational inactivation of the proapoptotic Bcl-2 homolog Bax. Nat Med 8: 274–281.

  19. Legarza K, Yang LX . (2006). New molecular mechanisms of action of camptothecin-type drugs. Anticancer Res 26: 3301–3305.

  20. Lu D, Wolfgang CD, Hai T . (2006). Activating transcription factor 3, a stress-inducible gene, suppresses Ras-stimulated tumorigenesis. J Biol Chem 281: 10473–10481.

  21. Miyazaki K, Inoue S, Yamada K, Watanabe M, Liu Q, Watanabe T et al. (2009). Differential usage of alternate promoters of the human stress response gene ATF3 in stress response and cancer cells. Nucleic Acids Res 37: 1438–1451.

  22. Morita S, Kojima T, Kitamura T . (2000). Plat-E: an efficient and stable system for transient packaging of retroviruses. Gene Ther 7: 1063–1066.

  23. Naka T, Sugamura K, Hylander BL, Widmer MB, Rustum YM, Repasky EA . (2002). Effects of tumor necrosis factor-related apoptosis-inducing ligand alone and in combination with chemotherapeutic agents on patients’ colon tumors grown in SCID mice. Cancer Res 62: 5800–5806.

  24. Nesterov A, Nikrad M, Johnson T, Kraft AS . (2004). Oncogenic Ras sensitizes normal human cells to tumor necrosis factor-alpha-related apoptosis-inducing ligand-induced apoptosis. Cancer Res 64: 3922–3927.

  25. Nicholson DW . (2000). From bench to clinic with apoptosis-based therapeutic agents. Nature 407: 810–816.

  26. Nobori K, Ito H, Tamamori-Adachi M, Adachi S, Ono Y, Kawauchi J et al. (2002). ATF3 inhibits doxorubicin-induced apoptosis in cardiac myocytes: a novel cardioprotective role of ATF3. J Mol Cell Cardiol 34: 1387–1397.

  27. Pan J, McEver RP . (1993). Characterization of the promoter for the human P-selectin gene. J Biol Chem 268: 22600–22608.

  28. Pelzer AE, Bektic J, Haag P, Berger AP, Pycha A, Schafer G et al. (2006). The expression of transcription factor activating transcription factor 3 in the human prostate and its regulation by androgen in prostate cancer. J Urol 175: 1517–1522.

  29. Pitti RM, Marsters SA, Ruppert S, Donahue CJ, Moore A, Ashkenazi A . (1996). Induction of apoptosis by Apo-2 ligand, a new member of the tumor necrosis factor cytokine family. J Biol Chem 271: 12687–12690.

  30. Ravasi T, Suzuki H, Cannistraci CV, Katayama S, Bajic VB, Tan K et al. (2010). An atlas of combinatorial transcriptional regulation in mouse and man. Cell 140: 744–752.

  31. Riley T, Sontag E, Chen P, Levine A . (2008). Transcriptional control of human p53-regulated genes. Nat Rev Mol Cell Biol 9: 402–412.

  32. Scott N, Hale A, Deakin M, Hand P, Adab FA, Hall C et al. (1998). A histopathological assessment of the response of rectal adenocarcinoma to combination chemo-radiotherapy: relationship to apoptotic activity, p53 and bcl-2 expression. Eur J Surg Oncol 24: 169–173.

  33. Sellers WR, Fisher DE . (1999). Apoptosis and cancer drug targeting. J Clin Invest 104: 1655–1661.

  34. Sheikh MS, Burns TF, Huang Y, Wu GS, Amundson S, Brooks KS et al. (1998). p53-dependent and -independent regulation of the death receptor KILLER/DR5 gene expression in response to genotoxic stress and tumor necrosis factor alpha. Cancer Res 58: 1593–1598.

  35. Sprick MR, Weigand MA, Rieser E, Rauch CT, Juo P, Blenis J et al. (2000). FADD/MORT1 and caspase-8 are recruited to TRAIL receptors 1 and 2 and are essential for apoptosis mediated by TRAIL receptor 2. Immunity 12: 599–609.

  36. Sussman RT, Ricci MS, Hart LS, Sun SY, El-Deiry WS . (2007). Chemotherapy-resistant side-population of colon cancer cells has a higher sensitivity to TRAIL than the non-SP, a higher expression of c-Myc and TRAIL-receptor DR4. Cancer Biol Ther 6: 1490–1495.

  37. Suzuki T, Fujisawa JI, Toita M, Yoshida M . (1993). The trans-activator tax of human T-cell leukemia virus type 1 (HTLV-1) interacts with cAMP-responsive element (CRE) binding and CRE modulator proteins that bind to the 21-base-pair enhancer of HTLV-1. Proc Natl Acad Sci USA 90: 610–614.

  38. Takimoto R, El-Deiry WS . (2000). Wild-type p53 transactivates the KILLER/DR5 gene through an intronic sequence-specific DNA-binding site. Oncogene 19: 1735–1743.

  39. Tamura K, Hua B, Adachi S, Guney I, Kawauchi J, Morioka M et al. (2005). Stress response gene ATF3 is a target of c-myc in serum-induced cell proliferation. Embo J 24: 2590–2601.

  40. Turchi L, Aberdam E, Mazure N, Pouyssegur J, Deckert M, Kitajima S et al. (2008). Hif-2alpha mediates UV-induced apoptosis through a novel ATF3-dependent death pathway. Cell Death Differ 15: 1472–1480.

  41. Wang J, Chun HJ, Wong W, Spencer DM, Lenardo MJ . (2001). Caspase-10 is an initiator caspase in death receptor signaling. Proc Natl Acad Sci USA 98: 13884–13888.

  42. Wang S, El-Deiry WS . (2003). Requirement of p53 targets in chemosensitization of colonic carcinoma to death ligand therapy. Proc Natl Acad Sci USA 100: 15095–15100.

  43. Wang Y, Engels IH, Knee DA, Nasoff M, Deveraux QL, Quon KC . (2004). Synthetic lethal targeting of MYC by activation of the DR5 death receptor pathway. Cancer Cell 5: 501–512.

  44. Wei CL, Wu Q, Vega VB, Chiu KP, Ng P, Zhang T et al. (2006). A global map of p53 transcription-factor binding sites in the human genome. Cell 124: 207–219.

  45. Wei MC, Lindsten T, Mootha VK, Weiler S, Gross A, Ashiya M et al. (2000). tBID, a membrane-targeted death ligand, oligomerizes BAK to release cytochrome c. Genes Dev 14: 2060–2071.

  46. Wiley SR, Schooley K, Smolak PJ, Din WS, Huang CP, Nicholl JK et al. (1995). Identification and characterization of a new member of the TNF family that induces apoptosis. Immunity 3: 673–682.

  47. Wu GS, Burns TF, McDonald III ER, Jiang W, Meng R, Krantz ID et al. (1997). KILLER/DR5 is a DNA damage-inducible p53-regulated death receptor gene. Nat Genet 17: 141–143.

  48. Wu X, Nguyen BC, Dziunycz P, Chang S, Brooks Y, Lefort K et al. (2010). Opposing roles for calcineurin and ATF3 in squamous skin cancer. Nature 465: 368–372.

  49. Xiang H, Fox JA, Totpal K, Aikawa M, Dupree K, Sinicropi D et al. (2002). Enhanced tumor killing by Apo2L/TRAIL and CPT-11 co-treatment is associated with p21 cleavage and differential regulation of Apo2L/TRAIL ligand and its receptors. Oncogene 21: 3611–3619.

  50. Yamaguchi H, Wang HG . (2004). CHOP is involved in endoplasmic reticulum stress-induced apoptosis by enhancing DR5 expression in human carcinoma cells. J Biol Chem 279: 45495–45502.

  51. Yan C, Lu D, Hai T, Boyd DD . (2005). Activating transcription factor 3, a stress sensor, activates p53 by blocking its ubiquitination. Embo J 24: 2425–2435.

  52. Yin X, Dewille JW, Hai T . (2008). A potential dichotomous role of ATF3, an adaptive-response gene, in cancer development. Oncogene 27: 2118–2127.

  53. Yoon K, Lee SO, Cho SD, Kim K, Khan S, Safe S . (2011). Activation of nuclear TR3 (Nr4a1) by a diindolylmethane analog induces apoptosis and proapoptotic genes in pancreatic cancer cells and tumors. Carcinogenesis 32: 836–842.

  54. Yoshida T, Maeda A, Tani N, Sakai T . (2001). Promoter structure and transcription initiation sites of the human death receptor 5/TRAIL-R2 gene. FEBS Lett 507: 381–385.

  55. Zhang C, Gao C, Kawauchi J, Hashimoto Y, Tsuchida N, Kitajima S . (2002). Transcriptional activation of the human stress-inducible transcriptional repressor ATF3 gene promoter by p53. Biochem Biophys Res Commun 297: 1302–1310.

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Acknowledgements

We thank Dr WS El-Deiry for a kind gift of valuable plasmid pGL2-Full. This work was supported in part by a grant-in-aid for Research on Priority Areas from the Ministry of Education, Culture, Sports, Science, and Technology of Japan [18012015, 18055008, 21590302] to SK and [22300330] to JM.

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Correspondence to S Kitajima.

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Supplementary Information accompanies the paper on the Oncogene website

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Taketani, K., Kawauchi, J., Tanaka-Okamoto, M. et al. Key role of ATF3 in p53-dependent DR5 induction upon DNA damage of human colon cancer cells. Oncogene 31, 2210–2221 (2012) doi:10.1038/onc.2011.397

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Keywords

  • DR5
  • ATF3
  • p53
  • CPT
  • TRAIL

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