Identification of SCN3B As a Novel P53-Inducible Proapoptotic Gene

Identiﬁcation of SCN3B as a novel p53-inducible proapoptotic gene

Katsuya Adachi1,3,5, Minoru Toyota1,2,5, Yasushi Sasaki1,5, Toshiharu Yamashita1, Setsuko Ishida1, Mutsumi Ohe-Toyota1, Reo Maruyama2, Yuji Hinoda2,4, Tsuyoshi Saito3, Kohzoh Imai2, Ryuichi Kudo3, Takashi Tokino*,1

1Department of Molecular Biology, Cancer Research Institute, Sapporo Medical University School of Medicine, S-1, W-17, Chuo-ku, Sapporo 060-8556, Japan; 2First Department of Internal Medicine, Sapporo Medical University School of Medicine, S-1, W-16, Chuo-ku, Sapporo 060-8543, Japan; 3Department of Obstetrics and Gynecology, Sapporo Medical University School of Medicine, S-1, W-16, Chuo-ku, Sapporo 060-8543, Japan; 4Division of Clinical Laboratory, Yamaguchi University School of Medicine, 1-1-1 Minami-Kogushi, Ube 755-8505, Japan

Tumor suppressor p53 is a transcription factor that ability to induce apoptosis is thought to be central to its induces growth arrest and/or apoptosis in response to tumor-suppressor activity. p53 is induced by many types cellular stress.To identify novel p53-inducible genes, we of stress, including DNA damage, oncogene activation, compared the expression of genes in normal mouse hypoxia, and loss of normal cell–cell contacts. It seems embryo fibroblasts (MEFs) to p53-null cells by cDNA likely that the activation of p53-dependent apoptosis can representational difference analysis.We report here that contribute to the inhibition of cancer development at expression of endogenous sodium channel subunit beta 3 several stages during tumorigenesis. (SCN3B) is upregulated in mouse embryonic fibroblasts p53 exerts various physiological functions through by DNA damage in a p53-dependent manner.In addition, transactivation of downstream genes. Among the p53- we found that SCN3B levels are upregulated in human inducible genes, induction of the cyclin-dependent kinase cancer cell lines by DNA damaging agents, as well as by inhibitor p21WAF1/CIP1 (hereafter referred to as p21) by p53 overexpression of p53, but not significantly by p63 or p73. is central to the activation of cell cycle arrest. However, Furthermore, we identified two putative p53-binding sites activation of p21 is not required for the induction of upstream of the first exon (RE1) and in the third intron apoptosis. Candidates for p53-regulated apoptotic tar- (RE2).The p53 protein can directly interact with the gets include mitochondrial proteins that are likely to putative p53-binding sites in vivo, as assessed by contribute to the release of cytochrome c from the chromatin immunoprecipitation.A reporter gene assay mitochondria, such as BAX (Miyashita and Reed, 1995), revealed that these two p53-binding sites are functional NOXA (Oda et al., 2000a), p53AIP1 (Oda et al., 2000b), response elements.The SCN3B protein appears to be PUMA (Nakano and Vousden, 2001; Yu et al., 2001) localized to the endoplasmic reticulum (ER).Introduction and also proteins that play a role in death receptor- of the SCN3B gene into T98G and Saos2 cells potently mediated apoptosis, such as Fas (Owen-Schaub et al., suppressed colony formation.Furthermore, we found that 1995), KILLER/DR5 (Wu et al., 1997), and PIDD (Lin adenovirus-mediated transfer of SCN3B induced apopto- et al., 2000). To date, there does not appear to be a single sis when combined with anticancer agents.The results gene that is the principal mediator of the p53 apoptotic presented here suggest that SCN3B mediates a p53- signal, and it seems likely that this response represents dependent apoptotic pathway and may be a candidate for the activation of multiple apoptosis-related genes. gene therapy combined with anticancer drugs. Identification of novel p53-inducible genes involved in Oncogene (2004) 23, 7791–7798. doi:10.1038/sj.onc.1208067 apoptosis is therefore important to better understand Published online 30 August 2004 the physiological functions of p53. To identify target genes induced by endogenous p53 under physiological Keywords: p53; SCN3B; apoptosis; ER conditions, we compared gene expression in embryonic fibroblasts from normal mice to that in p53-null (p53 (À/À)) mice by cDNA representational difference analysis (RDA). We report here the identification and Introduction characterization of the sodium channel subunit beta 3 (SCN3B) as a novel p53-inducible proapoptotic gene. Alterations of the p53 gene are among the most common and important events in human carcinogenesis (Hollstein et al., 1991). p53 inhibits cell growth through Results activation of both cell cycle arrest and apoptosis, and the Scn3b expression associated with p53 activity *Correspondence: T Tokino; E-mail: [email protected] 5These authors contributed equally to this work We used cDNA RDA to identify genes that are induced Received 21 January 2004; revised 27 July 2004; accepted 28 July 2004; by endogenous p53. Several cDNA fragments appeared published online 30 August 2004; to be differentially expressed in primary mouse embryo SCN3B is a mediator of p53-induced apoptosis K Adachi et al 7792 fibroblasts (MEFs) from p53(À/À) compared to MEFs by wild-type p53 in other rodents using REF-p53val135 from p53( þ / þ ) mice. The results from the cDNA containing a stably integrated murine temperature- RDA were confirmed by semiquantitative reverse sensitive mutant p53. RT–PCR analysis showed that transcription–polymerase chain reaction (RT–PCR). expression of Scn3b mRNA was detected at 311C but As shown in Figure 1a, expression of known p53- not at 371C (Figure 1c), demonstrating that the inducible genes identified by RDA, such as p21, cyclin G endogenous Scn3b gene is inducible in a rat embryo and Annexin 8, were dependent on p53 status and were fibroblast cell line by murine p53 in a wild-type induced by adriamycin which is known to induce conformation-dependent manner. endogenous p53, whereas Mdm2 was not significantly induced by adriamycin in MEFs. Among the cDNA Induction of SCN3B by p53 in human cancer cell lines fragments identified here, we focused on one clone that was not expressed in untreated p53( þ / þ ) MEFs, but From the human genome database we found that the was significantly induced by adriamycin in a p53- human SCN3B gene is composed of six exons and maps dependent manner (Figure 1a; Scn3b). Nucleotide to chromosome 11q23.3. To examine whether ectopic sequencing followed by a database search revealed that expression of the p53 family genes could increase this clone was identical to the mouse sodium channel expression of endogenous SCN3B mRNA in human Scn3b cDNA (Accession No. BC058636). Northern blot cells, human cancer cell lines were infected with each of analysis confirmed that Scn3b expression was induced in the p53 family genes by adenovirus-mediated transfer. MEFs following adriamycin treatment in a p53-depen- Replication-deficient adenoviral vectors harboring hu- dent manner (Figure 1b). man p53 (Ad-p53), p73b (Ad-p73b), p63g (Ad-p63g) If the Scn3b gene has an important role downstream genes, and the b-galactosidase gene (Ad-lacZ) as a of p53, it would be conserved across different species. control were infected into human cancer cell lines. We These predictions were tested in the following experi- used three human cancer cell lines, Saos2, H1299 and ments. We tested whether Scn3b expression is induced HCT116, that showed highly efficient gene transfer, with 90–100% of the cells staining for b-galactosidase activity at a multiplicity of infection (MOI) of 50–100 as described previously (Sasaki et al., 2002). Northern blot analysis revealed that SCN3B expression was induced by Ad-p53 in these three lines examined, especially highly in Saos2 cells (Figure 2a). In contrast, SCN3B mRNA was not significantly increased by either Ad- p73b or Ad-p63g in these three cell lines (Figure 2a). However, the more sensitive technique of semiquantitative RT–PCR revealed that SCN3B expression was increased by either Ad-p73b or Ad-p63g in Saos2 cells (data not shown).

Induction of SCN3B by DNA damage in human cancer cell lines Adenovirus-mediated transfer often employs exogenous gene expressed at levels likely to be higher than those ever encountered in vivo. To assess transcriptional induction of SCN3B at more physiological levels of p53, parental HCT116 cells with endogenous wild-type p53 and their derivative cells with targeted deletions of both p53 alleles p53(À/À) were treated with adriamycin. The induction of SCN3B was significantly detected Figure 1 Identification of p53 target genes by cDNA RDA. (a) in HCT116-p53( þ / þ ) but not in HCT116-p53(À/À) Semiquantitative RT–PCR analysis of Scn3b in MEFs of p53( þ / þ ) and p53(À/À) mice after treatment with 0.2 mg/ml adriamycin by Northern blot analysis in the condition which (ADR) for 24 h. RT–PCR analysis was performed as described in endogenous p53 and p21 were induced in HCT116- Materials and methods. We also performed semiquantitative RT– p53( þ / þ ) (Figure 2b). In addition, the induction of PCR analysis of known p53-target genes such as p21, cyclin G and SCN3B was detected in HCT116-p53( þ / þ ) but not in Annexin 8. GAPDHmRNA levels were examined as a control for HCT116-p53(À/À) after a 24 h treatment with etopo- integrity of the cDNA. (b) Northern blot analysis shows an elevation of Scn3b and p21 mRNA levels in MEFp53( þ / þ ) side (data not shown). Previous studies revealed that exposed to 0.2 mg/ml ADR for 24 h. Ethidium bromide staining of p73 protein is increased in cells exposed to chemother- 28S rRNA (28S) in the bottom row shows that equal amounts of apeutic agents through transcription and protein RNA were loaded in each lane. (c) Semiquantitative RT–PCR stabilization (Agami et al., 1999; Gong et al., 1999; analysis of rat Scn3b in REFp53val135 cells following thermoshift. These cells were incubated either at 371C (mutant-type p53 Yuan et al., 1999; Irwin et al., 2003; Sasaki et al., 2003). conformation) or at 311C (wild-type p53 conformation) for 16 However, we showed that the SCN3B gene is upregu- and 24 h lated by Ad-p53 but not significantly by Ad-p73b or

Oncogene SCN3B is a mediator of p53-induced apoptosis K Adachi et al 7793 extract from Ad-p53-infected and control cells. We then measured the abundance of candidate response elements within the immunoprecipitated complexes by PCR amplification. The ChIP assay revealed that p53 protein resided at DNA fragments containing RE1 or RE2 (Figure 3b, second and third rows, lanes 5 and 9). In contrast, other DNA fragments in the SCN3B promoter lacking RE1 or RE2 were amplified in the input chromatin-positive control for PCR but not in the immunoprecipitates with the anti-p53 antibody (e.g., SCN3B-PRO1 in Figure 3b, fourth row, lane 9). As expected, exogenous p53 protein resided at the p21 promoter containing a p53-response element in vivo (Figure 3b, first row, lane 9). These ChIP assay results suggest that the exogenous p53 protein is associated with RE1 and RE2 in vivo. Finally, we demonstrated by ChIP assay that endogenous p53 protein interacts with chromatin region containing its specific binding sites RE1 and RE2 in HCT116 cells treated with adriamycin (Figure 3b, lane 7). To determine whether the RE1 and RE2 sequences Figure 2 Induction of human endogenous SCN3B by p53. confer p53-dependent transcriptional activity, we per- (a) Northern blot analysis showing SCN3B mRNA induction after formed a heterologous promoter-reporter assay using a Ad-mediated transfer of the p53 gene family in human cancer cell luciferase vector prepared by cloning the oligonucleo- lines. Three human cancer cell lines were infected with adenoviruses tides corresponding to RE1 and RE2 upstream of a at an MOI of 50 or 100. In all, 10 mg of total RNA isolated 24 h minimal promoter (see ‘Materials and methods’). Con- after infection were loaded in each lane, and the same filter was rehybridized with human SCN3B and p21 cDNAs. Ethidium trol reporter plasmids pGL3-SCN3B-RE1mut and bromide staining of 28S rRNA (28S) in the bottom row shows that pGL3-SCN3B-RE2mut were generated by altering equal amounts of RNA were loaded in each lane. (b) Induction of potentially critical nucleotides of RE1 and RE2, SCN3B by DNA damage in HCT116-p53( þ / þ ) but not in respectively. H1299 cells were transiently cotransfected HCT116-p53(À/À) cells. Northern blot analysis was performed using total RNAs from cells untreated (À) or treated ( þ ) with with a reporter plasmid, together with a p53-expression 0.5 mg/ml adriamycin (ADR) for 24 h. Immunoblot analysis or control plasmid. By comparing the ratio of luciferase revealed that p53 and p21 protein were induced in HCT116- activity from the wild-type-responsive sequences p53( þ / þ ) following the ADR treatment (RE1wt and RE2wt) to the corresponding mutant- responsive sequences (RE1mut and RE2mut), we showed that when cotransfected with p53, luciferase Ad-p63g in several human cancer cell lines (e.g., activities from pGL3-SCN3B-RE1wt and pGL3- Figure 2a). Thus, these results lend further support to SCN3B-RE2wt are 50–100-fold higher than those from the notion that SCN3B induction is regulated by pGL3-SCN3B-RE1mut and pGL3-SCN3B-RE2mut, endogenous p53. respectively (Figure 3c). Together, these results also support the idea that both the RE1 and RE2 sequences p53-response elements in the SCN3B gene can mediate p53-dependent transcriptional activation, leading to the conclusion that SCN3B is a direct target To investigate whether the SCN3B gene is a direct target of p53. of transcriptional activation by p53, we searched for consensus p53-binding sequences in the human SCN3B Subcellular localization of the SCN3B protein gene. We found two potential p53-response elements, designated SCN3B-RE1 and SCN3B-RE2, (hereafter The PSORT II program predicted the presence of referred to as RE1 and RE2) which are located about potential endoplasmic reticulum (ER) targeting motifs 10 kb upstream of the first exon and in the third intron, in the SCN3B protein, suggesting that SCN3B might be respectively. RE1 (tGACTTGCTC tGcCTTGCCT) and an ER protein. To determine the subcellular localization RE2 (tGGCAAGgCT GAGCTAGTTC) sequences of SCN3B protein, we constructed a mammalian contain three- and two-mismatch nucleotides (lower expression vector encoding the full-length SCN3B letters) in noncritical positions within the 20-bp con- protein with an amino-terminal FLAG epitope. We sensus p53-binding sequences, respectively (Figure 2a, transiently transfected the plasmid into Saos2 cells and El-Deiry et al., 1992). To determine whether the p53 stained the cells using an anti-FLAG antibody. This protein can bind directly to the RE1 and RE2 sites in immunocytochemical analysis showed that the SCN3B vivo, we performed a chromatin immunoprecipitation protein is located in the cytoplasm and plasma (ChIP) assay using HCT116 cells infected with Ad-p53. membrane, and the signal was strongest around the Immunoprecipitation of DNA–protein complexes using nuclear membrane, a suspected ER-Golgi zone an antibody against p53 was performed on cross-linked (Figure 4, SCN3B). To determine the subcellular

Oncogene SCN3B is a mediator of p53-induced apoptosis K Adachi et al 7794

Figure 4 Subcellular localization of the SCN3B protein. Saos2 cells were transiently transfected with SCN3B expression vector, pCMVTag2-SCN3B, containing a FLAG epitope tag. Cells were ﬁxed and stained with antibodies to the FLAG epitope and to endogenous calnexin, an ER resident protein, followed by appropriate secondary antibodies. Transfected SCN3B (red) is colocalized with calnexin (green) in the ER. Nuclei were detected by staining cells with DAPI (blue). Original magniﬁcation, Â 1000

localization of the ectopic SCN3B protein, Saos2 cells were double stained with calnexin as an ER marker (Figure 4, calnexin). As shown by confocal microscopy, calnexin and SCN3B were colocalized in the ER (Figure 4, merge).

SCN3B enhances cellular sensitivity to anticancer agents To investigate whether SCN3B itself functions as a growth suppressor, we assayed the formation of geneticin-resistant colonies. An expression plasmid, pcDNA-SCN3B, containing the entire coding region of the human SCN3B cDNA was constructed using a mammalian expression vector pcDNA3.1( þ ), which harbors a gene conferring geneticin resistance. T98G and Saos2 cells were transfected with pcDNA-SCN3B Figure 3 The SCN3B gene is directly transactivated by p53. (a) and then grown in the presence of geneticin for 2 weeks. Human SCN3B gene structure and comparison of the two potential Figure 5 shows that introduction of the SCN3B gene p53-response elements (SCN3B-RE1 and SCN3B-RE2) to the p53- resulted in substantial growth suppression, as seen by a consensus-binding sequence (p53CBS). Nucleotides in capital letters represent a genomic sequence identical to the consensus, 3–10-fold decrease in the number of geneticin-resistant whereas lowercase letters identify disparities with the consensus. colonies, though not as complete as the growth The SCN3B-RE1 and SCN3B-RE2 sequences contain only three- suppression observed with p53. and two-mismatched nucleotides, compared with p53CBS. R, To investigate the proapoptotic effect of SCN3B, a purine; Y, pyrimidine; W, A or T. (b) ChIP assay showing that p53 protein binds to the SCN3B-RE1 and SCN3N-RE2 sites in vivo. recombinant adenoviral vector Ad-SCN3B expressing Immunoprecipitation of DNA–protein complexes using an anti- human SCN3B was constructed, and then Ad-SCN3B body against p53 (DO-7) was performed on cross-linked extract or a control Ad-lacZ vector were infected into Saos2, from untreated (no treat; lane 5), adriamycin-treated (ADR; lane 7), T98G and HCT116 cells. Flow cytometric analysis or Ad-p53 infected (lane 9) HCT116 cells. PCR amplification of performed at 72 h after infection showed a 2–3-fold in- the immunoprecipitated samples containing p21 promoter, pSCN3B-RE1, SCN3B-RE2, and SCN3B-PRO1 were performed crease in sub-G1 content, which is indicative of apopto- (first to fourth rows). Input chromatin (input), which represents a tic cells, by Ad-SCN3B relative to Ad-lacZ (Figure 6a). portion of the sonicated chromatin before immunoprecipitation SCN3B function may be an important mediator of were used as positive controls (lanes 2–4). Immunoprecipitates in cellular sensitivity to anticancer agents, since Scn3b the absence of antibody (Ab(À); lanes 6, 8 and 10) served as negative controls. DW, no template control (lane 1). The DNA is not expressed in untreated MEF-p53( þ / þ ), but is fragment containing the SCN3B-RE1 and SCN3B-RE2 were induced with adriamycin in a p53-dependent manner detected in the samples immunoprecipitated with the antibody (Figure 1a). To address this possibility, HCT116 cells against p53 in adriamycin-treated and Ad-p53-infected HCT116 were exposed to a low dose of anticancer agents cells (lanes 7 and 9). (c) Luciferase assay for the SCN3B-RE1 and following Ad-SCN3B infection. We observed the SCN3B-RE2 sequences. H1299 cells (p53 null) were cotransfected with pGL3-SCN3B-RE1, pGL3-SCN3B-RE2 or pGL3-p53CBS, enhancing effect of Ad-SCN3B on adriamycin- and together with expression vector pcDNA3.1-p53 (p53 þ )or etoposide-induced apoptosis (Figure 6b). In contrast, pcDNA3.1 (p53À). The relative luciferase activity was defined as Ad-SCN3B had less effect on cisplatin-induced apopto- the activity in the cells transfected with the pGL3-SCN3B-RE1wt sis. Thus, expression of the SCN3B gene induces cellular and pGL3-SCN3B-RE2wt divided by those from the pGL3- SCN3B-RE1mut and pGL3-SCN3B-RE2mut, respectively. The sensitivity to apoptosis by some but not all types of histograms represent the average of three independent experiments, drugs. In HCT116 cells, Ad-p53 infection did not and error bars show the standard deviation significantly result in apoptosis (Sasaki et al., 2001),

Oncogene SCN3B is a mediator of p53-induced apoptosis K Adachi et al 7795 Although a number of genes that are controlled directly by p53 have been identified, the SCN3B gene had not been identified in association with p53 induction. One reason may be that the expression level of SCN3B was still low in some types of cancer cells even after p53 overexpression (Figure 2a). This finding supports that the response to p53 expression varies considerably among different cell types. These results also suggest one potential basis for cell-type-specific responses to p53 and for pleiotropic biological effects of p53. The SCN3B gene encodes a polypeptide that is closely related in amino-acid sequence to the b1 subunit of a voltage-sensitive sodium channel (Morgan et al., 2000). The voltage-sensitive sodium channel confers electrical excitability on neurons, a functional property required for higher processes including cognition. The ion-conduction a subunit of the channel is regulated by two known auxiliary subunits, b1and b2 (Catterall, 2000). The b1 and b2 subunits of sodium channels have similar overall structures but are not closely related in amino-acid sequence. Both b subunits are unique among ion channel subunits in that they contain a large, glycosylated extracellular domain, a single transmembrane segment, and a small intracel- Figure 5 SCN3B suppresses the growth of human tumor cells. (a) lular domain, and appear to have dual functions G418-resistant colony formation assay of SCN3B-transfected – modulation of channel gating and cell–cell interac- human cancer cells. T98G (top) and Saos2 (bottom) cells were tion (Isom et al., 1992, 1995). However, little is known transfected with pcDNA3.1, pcDNA-SCN3B or pcDNA-p53, and then plated. Following 14 days of geneticin selection, cells were about the function of b3 subunit. Our immunofluor- fixed and stained with Giemsa. (b) The fraction of colonies (in escence data suggest that the transfected SCN3B percent) in each dish relative to the pcDNA3.1-transfected cells is protein is mainly detected in the ER (Figure 4), indicated. The vectors used for each transfection are shown below although it is possible that a small fraction of SCN3B the column protein is located in other cellular compartments. The subcellular localization of endogenous SCN3B protein should be examined when we obtain an anti-SCN3B but had enhancing effect on cisplatin-, adriamycin- and antibody in the future. The results here suggest that cisplatin-induced apoptosis (Figure 6b). SCN3B may be involved in a proapoptotic pathway through the ER. Recently, Bourden et al. (2002) identified Scotin, a novel p53-inducible proapoptotic protein located in the ER and the nuclear membrane, Discussion which is the first report to provide a link between p53 and the ER. We have shown that the induction of SCN3B by p53 is Intracellular organelles such as the mitochondria are conserved across multiple species. The endogenous key participants in apoptosis. However, the ER – where Scn3b gene was induced in mouse and rat embryo members of the BCL-2 family of proteins are also fibroblasts in a p53-dependent manner (Figure 1). In localized – also has been implicated in apoptosis addition, endogenous SCN3B was upregulated in (Scorrano et al., 2003). Furthermore, evidence is human cancer cells by ectopic p53 but not significantly emerging that the ER plays an important role in by p73b or p63g (Figure 2a). In response to exposure apoptosis (for a review see Ferri and Kroemer, 2001). to anticancer reagents, SCN3B was induced in human The ER can trigger cell signals to apoptosis in response cancer cells that possess wild-type p53 activity to ER stresses such as hypoxia, expression of viral (Figure 2b). We also identified two p53-response proteins, inhibition of glycosylation, and disruption of elements in the SCN3B gene (Figure 3). These results calcium storage in the ER (for a review see Kaufman, suggest that the SCN3B gene is a direct target of 1999). The release of calcium from the ER upon transcriptional activation by p53. Furthermore, trans- proapoptotic signaling or after drug treatment triggers duction of SCN3B enhanced apoptosis when combined the opening of the calcium-sensitive mitochondria with anticancer drugs in human tumor cells (Figure 6), permeability transition pore, allowing the release of which suggests that SCN3B transfer might permit a cytochrome c from the mitochondria to the cytosol. reduction in the dose of chemotherapeutic agents and Whether SCN3B is involved in promoting apoptosis in thereby minimize harmful side effects. response to ER stress remains to be determined. To identify genes whose expression is regulated by Therefore, further studies will determine whether muta- p53, several approaches were previously employed. tions of the SCN3B gene occur in tumors and whether

Oncogene SCN3B is a mediator of p53-induced apoptosis K Adachi et al 7796

Figure 6 The effect of adenovirus-mediated transfer of the SCN3B gene on apoptosis. (a) Ad-LacZ and Ad-SCN3B were infected into Saos2, T98G and HCT116 cells at an MOI of 50, 100 and 100, respectively. The sub-G1 content of each cell population was examined on a ﬂow cytometer 72 h after infection. (b) SCN3B sensitized human tumor cells to chemotherapeutic drugs. HCT116 cells were transfected with Ad-SCN3B, Ad-LacZ, Ad-p53 or mock treated (control) at an MOI of 100 followed by treatment with cisplatin (CDDP, 15 mg/ml), adriamycin (ADR, 0.2 mg/ml) or etoposide (VP16, 30 mg/ml) for 1 h. Representative histograms of DNA content 72 h after drug treatment are shown (left). The percentage of sub-G1 cells was determined by ﬂow cytometry 72 h after drug treatment (right)

the SCN3B is a bona fide tumor suppressor. In cDNA RDA conclusion, SCN3B displays the characteristics expected cDNA RDA was performed according to the published as one of the genes that can contribute to p53-dependent procedure with RNA isolated from MEF of p53( þ / þ ) mice apoptosis. The finding of SCN3B as a novel p53- and from MEFs of p53(À/À) mice. The nucleotide sequences inducible gene brings to light a potential role of the ER of the synthetic oligonucleotides used for the cDNA RDA in p53-dependent apoptosis. were as follows: RBgl-24, 50-AGCACTCTCCAGCCTCT CACCGCA-30; RBgl-12, 50-GATCTGCGGTGA-30; JBgl-24, 50-ACCGACGTCGACTATCCATGAACA-30; JBgl-12, 50- GATCTGTTCATG-30; NBgl-24, 50-AGGCAACTGTGC TATCCGAG GGAA-30; NBgl-12, 50-GATCTTCCCTCG-30. Materials and methods In all, 2 mg of double-stranded cDNA was digested with the restriction enzyme MboI, ligated to the R-Bgl-12/R-Bgl-24 Cell lines, cell culture and recombinant adenovirus adapter, and then amplified by PCR with R-Bgl-24 as a primer The human cancer cell lines used in this study were purchased to generate the ‘amplicon’. The adapter was removed from the from American Type Culture Collection (Manassas, VA, ‘amplicon’ derived from p53(À/À) MEFs by MboI digestion, USA) or the Japanese Collection of Research Bioresource and the resulting DNA was phenol extracted and ethanol (Osaka, Japan). All cell lines were cultured under conditions precipitated to generate the ‘driver’. The ‘amplicon’ prepared recommended by their depositors. The status of the endogen- from p53( þ / þ ) MEFs was gel purified and 2 mg was ligated ous p53 gene in these lines is wild type for HCT116, mutant for to a JBgl-12/JBgl-24 adapter to generate the ‘tester’. For the T98G, and p53 null for Saos2 and H1299. HCT116 p53 wild first subtractive hybridization, 500 ng of a JBgl-ligated tester type and its derivative HCT116-p53(À/À) lacking p53 were was mixed with 40 mg of the driver and then amplified by PCR kindly provided by Dr Bert Vogelstein (Howard Hudges with the JBgl-24 primer, resulting in the first difference product Medical Institute, Johns Hopkins University). MEFs from (DP1). DP1 was PCR amplified with the JBgl-12 and JBgl-24 p53( þ / þ ) mice and p53(À/À) mice were provided by Dr primers to convert both ends of the fragment to NBgl-12/ Motoya Katsuki (National Institute for Basic Biology, Japan). NBgl-24 adapters, and then the processes of subtractive A rat embryo fibroblast cell line, REF-p53val135, was hybridization and selective amplification (using alternately established by transforming with a murine temperature- the JBgl12 and JBgl24 adapters) were repeated to generate sensitive mutant p53val135 and ras (Morimoto et al., 2002). DP2. The molar ratios of the tester and driver in the reactions The generation and purification of replication-deficient producing DP2 were 1 : 400. The DP2 PCR products were recombinant adenoviruses containing p53, p73b, p63g genes cloned into the pCR4.0-T vector (Invitrogen) and sequenced. or the bacterial LacZ gene were described previously (Ishida The resulting sequences were compared with the GenBank et al., 2000; Sasaki et al., 2001). database using the BLAST search program.

Oncogene SCN3B is a mediator of p53-induced apoptosis K Adachi et al 7797 Semiquantitative RT–PCR 50-ACCTTTCACCATTCCCCTAC-30 (forward) and 50- GCCCAAGGACAAAATAGCCA-30 (reverse) for the p21 The cDNAs for gene-specific RT–PCR were synthesized using promoter, respectively (Shaulian et al., 2000). To ensure that random hexamers (Sasaki et al., 2000). The exponential phase PCR was performed in the linear range of amplification, of the amplification was determined between 27 and 32 cycles template DNA was amplified for a maximum of 32 cycles. to allow for semiquantitative comparisons among cDNAs PCR products from the ChIP assay were sequenced to verify from identical reactions. The PCR conditions involved an the identity of the amplified DNA. initial denaturation step at 941C for 1 min, followed by 32 cycles (for Scn3b) or 27 cycles (for mouse p21 and GAPDH) at 941C for 30 s – an optimal annealing temperature (601C) for Luciferase assay each primer set – and at 721C for 45 s. Oligonucleotide primer sequences were as follows: 50-CCTCCACTCTCAAT Reporter plasmids pGL3-SCN3B-RE1 and pGL3-SCN3B- TAACTGCACACCC-30 (forward) and 50-CCTGGTTAGC RE2 were constructed as follows: three tandem repeats of the GAGTCTCAGATCTTC-30 (reverse) for mouse Scn3b, 50- SCN3B-RE1wt (50-TGACTTGCTCTGCCTTGCCT-30) and TCACTGAAGAGGCGGGAGAAG-30 (forward) and 50- its mutant form SCN3B-RE1mut (50-TGAATTTCTCTG GAACAGAGTGAGGTTTAGTCCGTGTA-30 (reverse) for CATTTCCT-30), SCN3B-RE2wt (50-TGGCAAGGCT rat Scn3b, and 50-ACCACAGTCCATGCCATCAC-30 (for- GAGCTAGTTC-30) and its mutant form SCN3B-RE2mut ward) and 50-TCCACCACCCTGTTGCTGTA-30 (reverse) (50-TGGAAATGCTGAGATACTTC-30) were synthesized for GAPDH. The PCR products were resolved on a 3% and inserted upstream of a basal SV40 promoter in the nusieve agarose gel containing ethidium bromide. pGL3-promoter vector (Promega), and the resulting constructs were designated pGL3-SCN3B-RE1wt, pGL3-SCN3B-RE1- Northern blot analysis mut, pGL3-SCN3B-RE2wt, and pGL3-SCN3B-RE2mut, respectively. Cultures of H1299 cells at 50% confluence in In all, 10 mg of total RNAs were separated by electrophoresis 6 cm dishes were transfected with a reporter plasmid (1.5 mg), on a 1% agarose gel containing 2.2 M formaldehyde. The together with 1.5 mg of a pcDNA3.1( þ ) control vector integrity and equal loading of RNA in each lane were (Invitrogen) or a vector that expresses p53 (pcDNA-p53) confirmed by ethidium bromide staining of the gel. RNA was using Lipofectin (Gibco BRL, Rockville, MD, USA) as transferred onto a nitrocellulose transfer membrane (Protran; described previously (Sasaki et al., 2002). Cells were harvested Schleicher and Schuell Inc., Keene, NH, USA) by capillary 48 h after transfection for measurement of luciferase activity blotting in 20 Â SSC. Hybridization was performed with using the Luciferase Assay System (Promega). The ability to radiolabeled probes in 50% formamide, 5 Â Denhardt stimulate transcription was defined as the ratio of luciferase solution, 3 Â SSC, 0.1% SDS, and 100 mg/ml of salmon activity in the cells transfected with the pGL3-SCN3B-RE1wt sperm DNA at 421C overnight. After hybridization, the and pGL3-SCN3B-RE2wt relative to the activity in the cells membranes were washed in 1 Â SSC/0.1% SDS at room transfected with the nonresponsive reporter plasmids pGL3- temperature, followed by washes in 0.25 Â SSC/0.1% SDS at SCN3B-RE1mut and pGL3-SCN3B-RE2mut, respectively. As 601C. a control experiment, pGL3-p53CBS containing three copies of the consensus p53-binding sequence or its mutant form ChIP assay (Ishida et al., 2000) were transfected into H1299 cells with pcDNA-p53 or pcDNA3.1( þ ). All experiments were per- ChIP was performed using the ChIP Assay Kit (Upstate formed in triplicate and repeated on at least three independent Biotechnology) and an antibody against p53 (DO-7) as occasions. described (Morimoto et al., 2002; Sasaki et al., 2002). 2 Â 106 HCT116 cells were plated onto a 10-cm dish. These cells were infected with recombinant adenovirus Ad-p53 for 16 h or Immunofluorescence microscopy treated with 0.5 mg/ml of adriamycin for 24 h. Genomic DNA and protein were cross-linked by addition of formaldehyde The entire coding region of the human SCN3B cDNA was (1% final concentration) directly to culture medium and subcloned into mammalian expression vector pCMV-Tag2B incubated for 15 min at 371C. Cells were lysed in 200 ml of SDS (Stratagene) to place a FLAG epitope tag (DYKDDDDK) lysis buffer, and sonicated to generate 300–800 bp DNA in-frame at the N-terminal of SCN3B. The plasmid fragments. After centrifugation, the cleared supernatant was construct was sequenced and named pCMVTag2-SCN3B. diluted 10-fold with the ChIP dilution buffer and incubated The construct containing the epitope tagged cDNA was with the specific antibody at 41C for 16 h. Immune complexes transfected into Saos2 cells; 1 Â 104 of the cells that were precipitated, washed, and eluted as recommended. DNA– transiently expressed SCN3B were seeded into a four-well protein cross-links were reversed by heating at 651C for 5 h, slide chamber (Lab-tek chamber slide, Nalge Nunc Inter- and DNA was purified and resuspended in 50 ml of TE (10 mM nctiond, Naperville, IL, USA) and cultured for 24 h before Tris-HCl, 1 mM EDTA). In all, 5 ml of each sample were used immunocytochemical staining. Cells in the four-well slide as a template for PCR amplification. PCR amplifications of chambers were fixed in cold absolute acetone for 10 min. the SCN3B gene containing the putative p53-binding se- They were then incubated for 2 h with rabbit anti-FLAG quences (RE1 and RE2 described above) were performed on polyclonal antibody (5 mg/ml, Sigma-Aldrich) together with immunoprecipitated chromatin using the specific primers 50- mouse anticalnexin monoclonal antibody (1 : 1000 dilution, ACCCAGTAGGAGGTGATTGA-30 (forward) and 50-ACA BD Transduction Lab). The slides were then stained with TACCTGAGACTGGGCAA-30 (reverse) for RE1, and Alexa 488-conjugated goat anti-mouse IgG and Alexa 594- 50-CTCTGGTGTGAAATGAAGGG-30 (forward) and 50- conjugated goat anti-rabbit IgG (Molecular Probes) for 30 min, TCCTTCTGTTTTTACCCGAG-30 (reverse) for RE2, respec- and counterstained with 40,6-diamidino-2-phenylindole tively. PCR amplifications of SCN3B-PRO1 and p21 promoter (DAPI). After each incubation, unbound antibodies were were performed using oligonucleotides 50-TAGTCCTC removed by rinsing with PBS. The specimens were examined CAAACAGCACAG-30 (forward) and 50-GGAAGCT using a laser-scanning confocal microscope (Fluoview; TAACGCTGATATTC-30 (reverse) for SCN3B-PRO1, and Olympus, Japan).

Oncogene SCN3B is a mediator of p53-induced apoptosis K Adachi et al 7798 Assay for the formation of geneticin-resistant colonies At 48 h after plating, the cells were treated for 1 h with a low concentration of each drug (0.2 mg/ml adriamycin, 30 mg/ml An expression plasmid, pcDNA-SCN3B, containing the entire etoposide, or 15 mg/ml cisplatin), followed by incubation with coding region of human SCN3B cDNA was constructed using fresh, drug-free medium. For flow cytometric analysis at 72 h an expression vector pcDNA3.1( þ ), which harbors a gene after infection, cells were harvested by trypsinization and conferring geneticin resistance. Cells at 25% confluence in pelleted by centrifugation. Cells were then fixed in 90% cold 10 cm dishes were transfected individually with 5 mg of each ethanol, treated with RNase A (500 U/ml), and stained with expression plasmid (pcDNA-p53, pcDNA-SCN3B and propidium iodide (50 mg/ml). Samples were analysed on a pcDNA3.1 control vector) and 25 ml of Lipofectin (Gibco FACSCalibur flow cytometer (Becton-Dickinson, San Jose, BRL). At 24 h following transfection, cells were split 1 : 4 and CA, USA). Data were analysed using the ModFIT model grown in the presence of geneticin (Gibco BRL) (0.3–0.6 mg/ program (Becton-Dickinson). ml) for 14 days. The cells were fixed and stained with Giemsa, and the number of colonies was scored. Experiments were performed in triplicate and repeated on two independent Acknowledgements occasions. We are grateful to Dr Motoya Katsuki for MEFs from p53 nullizygote mice and Dr Bert Vogelstein for HCT116- p53(À/À) cell lines. We also thank Dr Joseph F. Costello for Flow cytometry valuable discussion in this study. This study was supported in Saos2, T98G, and HCT116 cells (2 Â 105) were plated in 6 cm part by Grants-in-Aid for Scientific Research on Priority Areas dishes. At 24 h after plating, the cells were infected with from the Ministry of Education, Culture, Sports, Science, and recombinant adenovirus, Ad-SCN3B, Ad-p53 or Ad-LacZ. Technology (MT, YH, KI and TT).

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